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Measuring the Conduction Velocity of Median Motor Nerve Axons

Introduction Electromyograhy (EMG) is a technique used to measure “changes in electrical potential that result from the conduction of action potentials along motor units during muscle contraction”1. EMG activity (measured in mV) is linearly related to the force produced by muscles2 and so it is used as a diagnostic procedure to assess pathology affecting the anterior horn cell, plexus, nerve roots, neuromuscular junction, peripheral nerve, and the muscle3. Recordings are made with an electrode which is either inserted directly into the muscle (needle electrode) or applied to the skin (extracellular electromyography using surface electrodes)4 and electrical activity picked up by the electrodes is displayed on an oscilloscope screen in the form of waves (EMG)5. An audio-amplifier is also used so that the activity can be heard as well as seen5. This set-up is known as an electromyography machine (fig 1). From the waveform output (graph), conduction velocity, amplitude (which depends on the synchrony and number of action potentials) and duration of action potentials (reflecting the degree of temporal dispersion) are key measurements for the diagnosis of neuromuscular disorders3. Electrical signals can be measured using a monopolar arrangement (i.e. measuring at only one site) or a bipolar electrode arrangement (i.e. measuring at two different sites) coupled with a differential amplifier to “increase the amplitude of the difference signal between the two recording electrodes with respect to the reference electrode”6.
Working of an EMG machine
All these recordings are made after a stimulating bar, having an anode (positively charged) and cathode (negatively charged) at a fixed distance, is placed on the subject and stimulates the selected nerve3. The nerve depolarizes (is stimulated) underneath the cathode, and the resulting action potential travels along the nerve in the customary physiological direction (orthodromically) and opposite to the physiological direction (antidromically)3. It is recorded using the two recording electrodes from the EMG machine which are attached to the muscle fibre3 using an electrode paste for low-impedance electrode contact7. These measure the electromagnetic field (EMF) that is generated by action potentials7. The potential voltage difference between the two separate electrodes can be calculated in order to provide information about the ability of that muscle to respond to stimulation7. To calculate nerve conduction velocity from an electromyogram the distance from the centre of the cathode also needs to be recorded.
In this experiment the nerve conduction velocity of the median motor nerve axons innervating the abductor pollicis brevis (APB) or thumb abductor muscles, as well as other muscles in the arm, forearm and hand was determined using extracellular (surface) electromyography. The median nerve was stimulated at the wrist and elbow and the electrical activity was recorded using electrode stickers and an isolated stimulator. Since the muscle was stimulated by depolarization of the median nerve at two distinct locations bipolar EMG was performed. Onset latency (explained below) and time taken from delivery of stimulus to recording in the EMG electrodes were measured and the nerve conduction velocity calculated.

The procedure used has been described below along with calculations involved in determining the nerve conduction velocity of median axons innervating the APB. Afterwards, the subject’s median motor nerve conduction velocity has been compared to the average velocity range.
Protocol and set-up To measure nerve conduction velocity of the median motor nerve the Evoked EMG and Nerve conduction velocity protocol from Kuracloud was followed8. An isolated stimulator and disposable ECG electrode stickers (recording electrodes) were used to collect the data inputted into the Kuracloud system. The recording electrodes were attached to the thumb and the ‘earth strap’ secured around the forearm as depicted in fig 1 and fig 2.
Checking equipment
Prior to beginning the experiment, the equipment was checked to ensure it was functioning correctly. The subject was asked to press their thumb and ring finger together as hard as they could, to see if any signal would be generated if the muscle was contracted. Two graphs were produced. Since no electrical activity was detected in the absence of muscle contraction, there was a flat line. Voluntary contraction resulted in voluntary an asynchronous burst of activity/staggered line around a constant voltage). Since electrical activity was detected only once muscle contraction began, it was concluded that the equipment was working correctly.

Measuring nerve conduction
First, the median nerve was located by dipping a bar in water and firmly pressing it on the subject’s wrist near their tendons of abductor pollicis longus. This was done to ensure the nerve was stimulated and the electrode did not move around during stimulation (fig 2). Pulses of 3.6mA were emitted from the stimulating bar on pressing “start”. At this point the subject experienced light tingling in their hand and all fingers. The amplitude was increased in order to produce a clear contraction of the APB muscle alone. The electrode was also moved laterally until only the thumb was twitching. The final position was marked on the subject’s skin with a pen (fig 3). Electrical activity from the APB muscle was recorded at the chosen amplitude and position (fig 4). The median nerve was also stimulated at the elbow as is done in bipolar EMGs. This eliminates electromagnetic noise that can interfere with the displayed wave. Other precautions to minimise noise included not having the mains cable and recording cable touch each other or any other bioelectric bodies. For this second recording the stimulating bar was placed on the lateral side of the tendon of the biceps bacchhii and the amplitude of ‘pulse’ was set to 11.2mA. Stronger external force was applied here as the nerve is deeper in this tissue. Once again, the electrode was shifted slightly until a clear stimulation of the subject’s APB muscle was observed i.e. the thumb twitched. The position was marked, and an electromyogram recorded (fig 5).
Fig 3: Points of stimulation marked on subject’s right arm with a pen.

Fig 4: Electromyograph of median motor nerve stimulated at the wrist. Start of action potential marked with orange line.
Data analysis and Results Fig 5: Electromyograph of median motor nerve stimulated at the elbow.
Nerve conduction velocity
Evoked EMG readings at the wrist
“The time interval between the stimulus at 0ms (can be confirmed by presence of stimulus artefact at 0ms) and the initiation of an evoked potential is known as onset latency”9. This is also the time it takes the impulse to travel from the stimulation point at the wrist to the recording electrode7 and “it reflects conduction speed along the fastest fibres”9. At the wrist it was recorded as 4.90ms. The point selected on the electromyogram can be seen (fig 4).
Additionally, the maximum amplitude of the subject’s response to median nerve stimulation at the wrist was recorded as 7.07mV in the Kuracloud program. Motor axon loss, a conduction block or incorrect electrode placement can result in low-amplitude of compound muscle action potential, apart from temporal dispersion (mentioned above).
Evoked EMG readings at the elbow
The onset latency of the subject’s median nerve stimulated at the elbow was recorded as 9.8ms and the amplitude was noted as 4.57mV (fig5) in Kuracloud.
Calculating nerve conduction velocity
An electromyogram shows the response of a particular muscle following nerve stimulation. This means that nerve latency and amplitude vary according to the point of nerve stimulation. Onset latency increases when the stimulation occurs farther away from the targeted muscle. However certain times do not change with stimulation location: the time taken for the current to reach the electrode and from there to stimulate the nerve, action potential propagation, time taken for neurotransmitters to be released, and the time for the electrical signal from the movement to be picked up by the equipment are the same at all stimulation locations. The distance between the marked stimulation sites (elbow and wrist), measured to be 320mm, was needed to calculate the median nerve conduction velocity of the subject. The nerve conduction velocity, calculated by diving the distance between the two sites by the difference in onset latencies at the elbow and wrist, was found to be 65.31m/s (see Table 1).
Table 1: Showing latencies at different points, which were used to calculate conduction velocity.
Discussion The electromyograms (fig 4, fig 5) show the depolarisation and repolarisation of APB muscle fibres as the muscle contracts and the thumb ‘twitches’ following stimulation from an electrode. From this the subject’s median motor nerve conduction velocity was determined to be 65.31m/s. While this value varies between individuals, the accepted range for the normal median motor nerve conduction in healthy individuals is49.48m/s – 66.92m/s10. There might be slight variation in this figure due to age or height, which are negatively associated with nerve conduction velocity10,11 , but not due to gender11. Variation may also occur because of temperature or compression7. The subject in this experiment was 165cm tall, 19 years old and tested in a room of approximately 22?C. Low temperatures decrease nerve conduction velocity. The subject did not note any feeling of compression, and so it is thought that this did not affect the results. Given the subject’s profile, their nerve conduction velocity appears to be higher than the mean velocity of others in their age group (see ‘Young adult’ under ‘Age group’ in Table 2), however it is still considered to be in the global normal range mentioned above. It would be valuable to compare this value to that of other subjects tested under the same conditions (same temperature) to accurately assess if their conduction velocity is notably different from the group mean. Further trials also need to be conducted to determine if this result is consistent. It is important to note, that the ‘normal range’ values may have been taken at a different temperature as most nerve conduction velocity studies are carried out at 33?C – 34?C12. Hence, it is recommended that during future trials, the subject be maintained at this temperature to ensure comparable results. If the subject’s results are consistently high it is likely that their nerves are well myelinated and the diameters of their axons larger than average, such that leakage is prevented, and resistance is decreased within axons.
Table 2: Showing normal conduction velocity in axons of median motor nerves10.
Nerve conduction tests are traditionally used to non-quantitively13 test for demyelination (caused by Guillan-Barrè syndrome) or axon loss (due to Friedrich’s ataxia) in large diameter axons, rather than the suspected pathology of the subject. This experiment tests the subjects median motor nerve; however, a variety of other peripheral nerves can also be tested: Sural sensory nerve, medial plantar nerve and the Ulnar sensory nerve14,15(for ‘normal’ velocities of different nerves see Table3).
Table 3: Showing normal nerve conduction velocities of different nerves in healthy individuals
In Guillan – Barrè syndrome (GBS) the body’s immune system attacks parts of the peripheral nervous system and damages the myelin sheath surrounding axons. As a result, axons are exposed to damage16 and saltatory conduction is prevented, slowing down nerve conduction velocity. This causes muscle weakness and possible sensation changes as damaged nerves might pass on abnormal sensory signals from the body16. Studies have shown GBS to result in decreased amplitude and nerve conduction velocity17. This fall in amplitude is characteristic of a sub-type of GBS: acute motor axonal neuropathy, which is characterized by the immune system attacking the node of Ranvier instead of the Schwann cells making up the myelin sheath17.
Friedrich’s ataxia is an inherited disease which causes progressive nervous system damage which results in impaired muscle coordination18. The degradation of neuronal cell bodies and axons can be detected by a significant decrease in amplitude identified during nerve conduction studies. The nerve conduction velocity remains unchanged19.
References 1 Hess, U. (2009). Facial EMG. In E. Harmon-Jones

Evolution of Pregnancy

The Evolution of Pregnancy
325Mya amniotes diverged into two distinct lineages; diapsids and primitive mammals(Brawand et al. 2008), which is believed to have occurred due to different environmental factors with few similarities surviving today. The mammalian lineage further spit into the prototherian mammals (monotremes) and the therian mammals (eutherians and marsupials) approximately 179Mya (Brawand et al. 2008), with the main reproductive differentiation being the extent of oviparous and viviparous nature of the two individual lineages (Rothchild 2002).
Interestingly, the vitellogenin gene that was found to be present in the amniote is responsible for being the precursor for the proteins that make up the composition of the yolk (Babin 2008) found in oviparous animal’s eggs. This gene began with two counterparts VIT1 and VITanc (Brawand et al. 2008) 350Mya before the divergence of the amniotes and through evolution it was determined by (Menkhorst et al. 2009; Kin et al. 2014) that the VITanc duplicated into VIT2 and VIT3 along the mammal lineage. The functionality of the loss of VIT3 gene 179Mya could be the reason for the split into the prototherian and therian lineages as it could have given rise to the viviparious nature of the therians. Furthermore, within this therian lineage there was a loss of the VIT1 independently for both the eutherian and metatherian lineages (Brawand et al. 2008), hence could indicate why the eutherians are more reproductively differentiated than the metatherians (marsupials) in comparison to the early amniotes (Rothchild 2002).
Additionally, the decreasing reliance on vitellogenin could be attributed to the elaboration of the placenta (Vincent et al. 2015), an organ that develops in the uterus during pregnancy that functions to provide oxygen and nutrients to the fetus (Wagner et al. 2014). There is uncertainty surrounding the reasons as to why this structure originally developed, however it could be due to the increase in survivability of the young through viviparious birth, hence making viviparity the evolutionary advantage. This is supported by Rothchild’s (2002) statement “viviarity has appeared in many forms among many members of every vertebrate class except brids, and that virtually every case of similarity is proably die to convergence or parallelism than of an evolutionary trend”.
Regardless of why, the endometrial stromal cells required for the placental production were believed to be present in therian mammals prior to their divergence (Vincent et al. 2015). The eutherian mammals have evolved to undergo decidualization (Lynch et al. 2011), the process of remodelling of endometrial stromal cells during pregnancy or in some species as part of their normal sexual cycle, which produces the placenta and many other traits specialised for prolonged pregnancy (131day mean) (Vincent et al. 2015), explaining the loss the shell and yolk characteristics. Contrarily, marsupials also have a type of placenta in the absence of decidualisation (Wagner et al. 2014), however it is formed by the yolk sac which only allows the fetus to attach for a limited time during pregnancy (Menkhorst et al. 2009), resulting in a shorter gestation period (25day mean) (Vincent et al. 2015). Contrarily, Babin (2008) determined that monotremes lack the ability to form a placenta, hence reflecting their oviparous nature.
Furthermore, in contrast to the eutherian mammals, oviparity is still present in the marsupial and monotreme mammals (Roberts et al. 2016), therefore still utilise an egg coating, which is thought to be a left-over trait of reptiles (Guillette 1993), persisting traditionally until hatching in the monotremes. In marsupials, as they also have a placenta, the conceptuses are enclosed in the shell coat as the zygote passes through the utero junction and inter the uterus where the coat deposition continues until the somite stage (Menkhorst et al. 2009;Renfree 2010), hence shed before birth. Additionally, the marsupials only have white yolk within their eggs (Roberts et al. 2016)
that functions to provide the extra-celluar matrix that contains hyaluronon stabilizing proteins for epithelial construction (Menkhorst et al. 2009) indicating they can now rely on the placenta for nutrients therefore permitting the loss of yellow yolk.
Unlike the marsupials, monotremes, specifically the platypus, have been found to contain a VIT2 3 hybrid gene that is thought to have evolved under selective pressures (Brawand et al. 2008; Lynch et al. 2008), keeping the most important coding so that the egg can be reduced in size but is still functional for birth. The eggshell consists of three parts; the white yolk, the germinal plasma and the yellow yolk (Menkhorst et al. 2009), which functions to provide nutrients in the form of protein, lipids and carbohydrates (usually glycogen) (Wildman et al. 2006). The yellow yolk is believed to have decrease substantially from the first amniotes due to the loss of vitellogenin1 and the ability to provide most of the nutrients for the young through lactation after pregnancy. This is supported by the presence of caseins in the milk of monotremes, a protein which emerged in the common mammalian ancestor and has VIT-like functionality by providing essential amino acids and binds calcium and phosphorus, required for skeletal growth, to the young (Brawand et al. 2008). Lactation originated from the simple egg-wetting function in ancestral mammals but evolved toward a new nourishment resource, developing the presence of casein, therefore permitting the Vitellogenin gene reduction Renfree 2010hjgfv.
Furthermore, monotremes are at the base of the mammalian tree which offers a unique opportunity to understand major aspects of mammalian genome evolution (Menkhorst et al. 2009). This is specifically interesting when observing their oviparous nature and their ability to lactate as this is considered to be the intermediately state of evolution from egg laying to eutherian viviparity (Wagner et al. 2014), however their mechanisms of functionality are relatively unknown. The echidna, a monotreme, is to be studied to provide insight into these intermediately pregnancy characterises and their behaviour to store their egg in a posterior facing pouch until hatching as this foreshadows that of the marsupials, who keep their young In the pouch for extended periods of time (Wagner et al. 2014). These individual behaviours of the echidna could provide further insight into the evolutionary differences between the prototherian and therian lineages and also the divergence of the amniote 350Mya.
References:
Babin, P.J. (2008) Conservation of a vitellogenin gene cluster in oviparous vertebrates and identification of its traces in the platypus genome. 413(1-2), 76-82
Brawand, D., Wahli, W., and Kaessmann, H. (2008) Loss of Egg Yolk Genes in Mammals and the Origin of Lactation and Placentation. 6(3), e63
Guillette, L.J., Jr. (1993) The Evolution of Viviparity in Lizards: Ecological, anatomical, and physiological correlates lead to new hypotheses. BioScience 43(11), 742-750
Kin, K., Maziarz, J., and Wagner, G.P. (2014) Immunohistological Study of the Endometrial Stromal Fibroblasts in the Opossum, Monodelphis domestica: Evidence for Homology with Eutherian Stromal Fibroblasts. 90(5), 111-111
Lynch, V.J., Leclerc, R.D., May, G., and Wagner, G.P. (2011) Transposon-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals. Nature Genetics 43, 1154
Lynch, V.J., Tanzer, A., Wang, Y., Leung, F.C., Gellersen, B., Emera, D., and Wagner, G.P. (2008) Adaptive changes in the transcription factor HoxA-11 are essential for the evolution of pregnancy in mammals. Proceedings of the National Academy of Sciences 105(39), 14928-14933
Menkhorst, E., Nation, A., Cui, S., and Selwood, L. (2009) Evolution of the shell coat and yolk in amniotes: a marsupial perspective. 312B(6), 625-638
Renfree, M.B. (2010) Review: Marsupials: Placental Mammals with a Difference. 31, S21-S26
Roberts, R.M., Green, J.A., and Schulz, L.C. (2016) The evolution of the placenta. Reproduction (Cambridge, England) 152(5), R179-R189
Rothchild, I. (2002) The Yolkless Egg and the Evolution of Eutherian Viviparity. 68(2), 337-357
Vincent, Mauris, Kapusta, A., Brayer, K., Silvia, Erik, Emera, D., Shehzad, Grützner, F., Bauersachs, S., Graf, A., Steven, Jason, Francesco, Feschotte, C., and Günter (2015) Ancient Transposable Elements Transformed the Uterine Regulatory Landscape and Transcriptome during the Evolution of Mammalian Pregnancy. Cell Reports 10(4), 551-561
Wagner, G.P., Kin, K., Muglia, L., and Pavli, M. (2014) Evolution of mammalian pregnancy and the origin of the decidual stromal cell. The International Journal of Developmental Biology 58(2-3-4), 117-126
Wildman, D.E., Chen, C., Erez, O., Grossman, L.I., Goodman, M., and Romero, R. (2006) Evolution of the mammalian placenta revealed by phylogenetic analysis. Proceedings of the National Academy of Sciences 103(9), 3203-3208
(Renfree 2010)

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