Human nerve fiber microelectrode recording experiment
Human nerve fiber microelectrode recording experiment
The purpose of this chapter is first to introduce the technique to scientists who wish to use it in a practical way. Secondly, typical examples of the results obtained with this technique, such as the role of musculocutaneous afferents in kinesthesia (see later in the results section), will be discussed in more detail. The role of other afferents in kinesthesia has also been studied.
Operation method
Experiments on microelectrode recording technology for human nerve fibers
Materials and Instruments
Subjects Move move As mentioned earlier, the purpose of this chapter is to provide a how-to guide for nerve fiber microelectrode recording techniques. In the following, we will give examples of techniques used in our laboratory to record the M nerve. Although many of the situations mentioned below are common and effective for recording other nerves, it is inevitable that some situations are only specifically applicable to our laboratory equipment. The experiment should be conducted in a shielded room, providing a pleasant atmosphere for the subject, with relevant light, sound and color. Also the experimental equipment should be concealed as much as possible. Before the experiment begins, have the subject sit comfortably in an armchair (Fig. 30-4). The legs are placed in a padded groove so that they can be maintained in a relaxed state without any muscular activity. The knee is flexed at approximately 120 to 130°. The right foot is placed on a stationary flat surface, and the left foot is secured to a rotatable pedal, the axis of which is anterior to the left ankle. The rotatable pedal is connected to an electromechanical motor whose movement parameters (e.g., speed, amplitude) can be adjusted and controlled. The signals recorded by the microelectrodes are amplified and output to an oscilloscope and a loudspeaker (Fig. 30-4), and neural activity is continuously monitored at different stages throughout the experiment. To record active or passive movements of the joints (dorsiflexion or plantarflexion), the subject is fitted with an additional system consisting of two lightweight, rotatable rods, one taped to the subject's leg and the other to the foot. Any rotation that occurs between the two rods, centered on the epicondyle, can be recorded with a linear potentiometer. The skin around the electrode insertion point is thoroughly cleaned with antibacterial soap in consideration of sterility and skin softening. Standard EMG surface electrodes (refer to Chapter 26 for details) were placed on the muscle associated with the experimental observation. Prior to the start of the recording, the subject should undergo some training, such as performing some casual movements as required by the experimental design, but without 'interfering' with the overall muscle activity. Avoiding such disturbances during nerve fiber microelectrode recording is essential for two reasons: first, they may cause displacement of the electrodes, resulting in loss of recording units or, worse, nerve damage. Second, even small EMG activity in muscles near the electrodes may cause distortion of the neural signals induced and recorded by motor unit activity. 1. Palpate the nerve. The first task is to locate the nerve alignment precisely and mark the skin surface with a pen. If the nerve course is too deep or too shallow, its depth can be changed by gently extending and flexing the knee joint. 2. Insertion of electrodes. The microelectrode is slowly inserted transdermally by hand through alternating gentle pushes and relaxations. When the electrode penetrates the skin, it is recommended to relax for one minute to observe the subject's response. With all these steps, despite great care, it is possible for the subject to be affected by the experimental or laboratory environment and to experience fainting. Therefore, it is important to pay close attention to the subject's state. Things like head scratching, frequent yawning, heat sensations, and sweating are warning signs. If these signals appear, the experiment should be stopped immediately. 3. Approach the nerve through the subcutaneous tissue. Approach the nerve using the same push-push method described above. The time required for this procedure varies from experiment to experiment. Sometimes it takes only a few minutes, but usually it takes about an hour. To guide the electrode tip into the nerve, many research groups are applying weak electrical impulses (1 to 6 V, wave width 0.2 ms) through the recording electrode to induce abnormal sensations in the subject or localized muscle contractions in the range of the muscle bundles. However, there are some problems with this approach. Psychologically, the fact that electrical stimulation will be used in the experiment can be frightening to some subjects and should therefore be avoided. In addition, in our experience, inappropriately applied electrical stimulation may cause strong motor nerve activation, i.e., the 'startle response', and may therefore both affect the stability of the recordings and jeopardize the subject's sense of security. In any case, when the electrodes are close to the nerves, keep asking the subject how he/she feels. This information can give the experimenter important clues for deciding whether to maintain or change the direction of the electrode impingement. For example, if the subject reports a "deep and diffuse" pain sensation, this suggests that the electrode is being advanced toward the tendon. If this occurs, or as soon as the subject feels any kind of pain, stop or even slightly back off the electrode. Usually the pain will go away at this point. Alternatively, if the subject feels a sense of touch due to mechanical stimulation of the nerve and is able to localize it easily, the experimenter can thus determine that the electrode is entering in the right direction. It should be emphasized here that the subject is told to name the sensory projection area and not to use it for pointing (as this may induce a great deal of muscular activity). 4. Reaching the nerve. When the electrode reaches the reaching nerve, a multi-unit discharge can usually be heard through the loudspeaker. It is important to find the correct unit within the nerve at this point. This unit can only be accurately identified when the electrode is 'free-floating' and the individual units are separated and stabilized. The precise identification process will be described separately later (see "Identification of Fibers"). However, before trying to stabilize the recording and make the electrode 'free-floating', a preliminary identification should be made. For this purpose, it is useful to continually tap the skin in the innervated area of the nerve, or to press on a tendon, muscle belly, or mildly passively pull the muscle innervated by the nerve. The experimenter can crudely identify any recorded afferent fibers and the type and site of their receptors by such stimulation. Figure 30-5 illustrates the detailed flow of this initial identification process. As early as the electrode reaches and enters the nerve, the spontaneous activity of the fiber may suggest its receptor origin. At the instant the electrode reaches the nerve, many fibers have a high-frequency discharge. If this response is completely quiescent within Bu 2s, it is mostly skin afferent. If this response is not completely quiescent and there is a sustained spontaneous discharge, it is probably afferent from intramuscular receptors. If the spontaneous discharges showed regular clustered bursts, they were probably efferent fibers from the vegetative nerves. 5. Stable recording. Finally, when the electrode records a signal from a single neuron, this signal must be isolated and stabilized. This can be accomplished by making tiny advances of the guiding electrode based on the monitor sound produced by successive stimulation of the afferent nerve. The main problem at this stage is that when the experimenter releases the electrode and allows it to 'free float' within the tissue, the elasticity of the skin usually pulls the electrode out slightly, making it easy to lose the signal. Even if some sort of micromanipulator could be attempted at this stage, such an attempt may not be useful due to mechanical resistance and the fickle nature of skin elasticity. Moreover, any attempt to immobilize the electrodes in this way could potentially damage the nerves in the unlikely event of sudden movement or involuntary activity by the subject. At this stage, a more precise identification of the recording fibers is still necessary, even though the experimenter has a basic grasp of the type of unit when the recording is stable. Unlike animal experiments, measuring the conduction velocity of nerve fibers in humans is not an easy task. Therefore, the recorded fibers need to be classified according to their nature, and those fibers that are difficult to classify should be abandoned. Indeed, there are problems with this qualitative classification, which is why research papers applying microelectrode recording techniques to nerve fibers often include statements such as, "These afferent fibers may be identified as ……" Burke discussed the problems associated with qualitative classification of nerve fibers in his 1997 paper. A more detailed account of this classification can also be found in the work of Edin and Vallbo (1990). It is important to note that it is unrealistic to detect every fiber using the identification methods presented in the different charts, since recorded fibers are hardly stable for a sufficiently long period of time. This requires the experimenter to choose an appropriate identification method according to the purpose and requirements of the experiment at hand. However, it is very important that the precise criteria used to characterize fibers are clearly stated in the study. Making the distinction between muscle-tendon afferents requires a series of physiologic tests (see Table 30-1). Of course, some generalized assessments are worth mentioning. Muscle tendon afferents are divided into three categories: primary musculocutaneous afferents distributed at the end of the primary musculocutaneous tract (primary MSA); secondary musculocutaneous afferents distributed at the end of the secondary musculocutaneous tract (secondary MSA), and Golgi tendon organ afferents distributed at the Golgi tendon organ (GTO afferents). All types of afferents can be activated by pressing the tendon of the muscle that hosts the receptor. Nonetheless, the pressure required to activate primary and secondary musculocutaneous afferents is much lower than the pressure required to activate Golgi tendon organ afferents. In addition, Golgi tendon organ afferents are highly sensitive to muscle contractions activated at low intensities. In terms of instantaneous frequency, they responded fairly regularly during casual contractions that were maintained at low levels, and the instantaneous frequency increased progressively with the degree of contraction, which is likely to be related to the continuous incorporation of new motor units. In the vast majority of If cases, a small, blunt object (e.g., the upper end of a pen) that can be pressed against the muscle belly or tendon to probe the site that elicits the maximal response can help us to localize the receptors in the afferent fibers. Single contraction test: a single isometric contraction elicited by transcutaneous electrical stimulation of a muscle nerve. Rapid diastolic burst: a slowly increasing random contraction followed by a single abrupt diastolic detraction Sensitization: repeated rapid detractions followed by holding the muscle in one long or short state for several seconds. Subsequently, its response to slow variable-rate detraction was recorded. For passive detraction sensitization, the intensity of the response to a slow variable-speed pull after the muscle has been held in a shorter state should be greater than the intensity of the response after the muscle has been held in a longer state. Some of the physiological and morphological differences between the different types of cutaneous receptors are shown in Table 30-2 (see Vallbo and Johansson 1984; Johansson and Vallbo 1983 for details). There are four main types of tactile units: slow-adapting (SA) and fast-adapting (FA), which can be subdivided into two subtypes (Type I and Type II) based on receptive field properties. In general, these afferent fibers have no spontaneous activity. Similar to GTO afferents, these cutaneous afferents are transiently activated when the electrode enters the nerve, and the issuance ceases in the following seconds. Therefore, in order to stabilize the recording and identify the receptive field' it is necessary to continuously touch the skin in the nerve distribution area. As a preliminary identification, applying a tactile stimulus to the skin surface (e.g., tapping the skin, Fig. 30-5) can identify fast-adapting afferents. Fixed or dynamic application of pressure to deeper sites activates slow-adapting receptors (deep pressure, Fig. 30-5). Identification of joint afferents can be difficult. Even so, it is possible to identify afferent nerves distributed across a joint receptor according to the following criteria. A pair of taps on the skin does not respond A pair of presses on neighboring muscles does not respond A pair that responded to sustained pressure on the joint capsule, but not on the adjacent bone Injurious afferents squeezing the skin, pinprick sensory fields, or warm stimulation all activated multisensory C fibers. Thermal stimulation can be performed with a radiant heat source or with a commercially available temperature stimulator (SomedicAB, Stockholm). Application of nerve fiber microelectrode recording techniques also allows recording of a, 7 and sympathetic efferent fiber activity. Identification of a and 7 motor neurons is more complex, but identification of sympathetic efferents is fairly easy. The differences in physiologic properties between these motoneurons are shown in Table 30-3 (see Ribot et al. 1986 for details). Multi-unit sympathetic activity is easily recognized in the form of brief clustered discharges. The following is a description of multi-unit sympathetic activity by Delius and his collaborators (1972) A subject with clustered discharges visible in the relaxed state, not accompanied by EMG activity I Irregularity in the sequence of discharges, synchronized with cardiac beats, thus showing a typical temporal phase pattern the average frequency of the cluster discharges is lower than that of the mechanosensory afferents. Application of nerve fiber microelectrode recording techniques also allows recording of individual units of sympathetic activity. For example, Hallin,Torebjdrk have recorded such single units of sympathetic activity on intact cutaneous nerves. The criteria they proposed for identifying single units of sympathetic activity are as follows (see Hallin and Torebjdrk1974 for details). One is associated with group sympathetic activity. The activity of a single unit usually occurs in conjunction with a group clustered discharge as described above. — no clear receptive field. Stimulation from any part of the body can activate these fibers Latency of a reflex response. Reflex latencies to different types of stimuli are relatively long (>0.5 seconds) Lidocaine (1%) given distal to the point of recording does not block its activity Related to Galvanic Skin Response The results shown in this chapter will illustrate how nerve fiber microelectrode recording techniques can be used in the study of the neurosensory mechanisms of human motion sensation (proprioception). It is generally accepted that proprioception is a composite sensation in which the simultaneous activation of many different types of peripheral receptors (e.g., joint, skin, and muscle receptors), together with central signals from the motor commands themselves, creates a sensation of position and movement. Feedback from intramuscular receptors is quite important for proprioception. For example, a mechanical vibration applied to a tendon of a limb can induce the illusion that the limb is moving. Indeed, vibration can always elicit the illusion of movement consistent with the sensation that the vibrated muscle or muscle group is being elongated (Goodwin et al. 1972; Roll and Vedel 1982). At this time, the general explanation for these movement illusions was that vibration preferentially activated tendon receptors, properties that had previously been described only in cats. With the development of microelectrode recording techniques for nerve fibers, it has become possible to accurately study afferent information from the localization of the appearance of the illusion. Moreover, some of the following questions can be stated more precisely: ① Which tendon receptors (primary or secondary MSAs or GTO afferents) are actually activated by vibration? (ii) What is the pattern between stimulus parameters and afferent activity? (iii) Finally, by recognizing the precise composition of the afferent information recorded during a specific occurrence of a particular movement, one can construct a vibratory pattern that can be used to generate an afferent message that is as similar as possible to the real one. In this case, is not the connection between the vibration-induced motion illusion and the actual motion performed clear? Using nerve fiber microelectrode recording techniques, it was found that primary MSAs were highly sensitive to mechanically vibrating tendons (vibrations with peak spacing of 0.25 to 0.5 mm applied to a resting muscle; Burke et al. 1976; Roll and Vedel 1982; Roll et al. 1989), whereas secondary MSAs and GTO afferents showed only moderate sensitivity to vibration degree of sensitivity (Fig. 30-6B). For primary MSAs, a one-to-one stimulus-response relationship exists in the frequency range of 1-100 Hz (Fig. 30-6B). This means that by adjusting the vibration frequency, the discharge frequency of the primary MSA can be varied proportionally (Fig. 30-6A). Thus, the human nerve fiber microelectrode recording technique demonstrates that vibratory stimuli applied to the tendon can activate the primary MSA pathway highly selectively. This precise one-to-one relationship between vibration frequency and afferent response frequency makes tendon vibration a very useful tool in the study of the perceptual properties of proprioceptive information. These studies have revealed some of the characteristics of afferent information during natural movement (top panel of Fig. 30-6A and Fig. 30-7). — Primary MSA activates rapidly at the onset of movement I Its discharge frequency increases continuously during homogeneous locomotion At the end of the movement, the discharge frequency returns to a steady state depending on the position reached — passive detractor muscles, i.e., the lifting resistors of the muscle performing the primary movement, have enhanced discharge activity These properties can thus be used to study the construction and functional use of vibratory modes, allowing the nature of the illusion to be elucidated. For example, the construction of a vibratory pattern applied to an extensor muscle and which can mimic the general characteristics of the uniform flexion illusion described above (Fig. 30-7B). Indeed, further simulation of the primary MSA response using nerve fiber microelectrode recording techniques has the potential to use even more complex vibratory modes, such as multiple vibrators that can be applied to the four muscle groups of the wrist (Fig. 30-8A). By varying the frequency of vibration, the duration of action of each stimulus and the time of vibrator initiation, and by applying multiple vibrators simultaneously or sequentially (Fig. 30-8B), more complex motion illusions can be produced, including several linear geometries (Fig. 30-8C) (Roll and Gilhodes 1995). Although nerve fiber microelectrode recording technology has traditionally been used to study the most basic neurophysiological functions of movement and sensation, the above results suggest that this technology can also be used to study more advanced integrative brain functions, revealing cognitive processes such as memory and recognition of movement forms in humans. For more product details, please visit Aladdin Scientific website.
Tungsten electrode Concentric round electrode
