**4. Conditioning of the evoked phasic ("synchronous") muscle activity**

So far, we have discussed the case of asynchronous voluntary activity of MUs that generates the EMG interference pattern. Another way to assess spinal cord processes is by means of reflex-generated compound muscle action potentials (CMAP).

A variety of reflexes (stretch reflex, cutaneous reflex, H-reflex, etc) has been studied at rest, during locomotion and during the performance of a number of motor tasks in an attempt to better understand how the central nervous system (CNS) integrates the descending signals with those coming from the periphery [1]. Ascending signals from the periphery are incorpo‐ rated into motor plans in order to continuously update the CNS and generate suitable commands to muscles that will work in concert to produce a functionally relevant motor output. At the spinal cord level, the afferent influx coming from muscles, joints and skin help to sculpt motor behavior by playing a significant role in the modulation of the excitability of different reflex pathways [1].

The stretch reflex pathway is one of special interest and will be the focus of this topic. The excitability of this pathway (or parts of it) can be assessed by means of either electrical stimulation of peripheral nerve (H-reflex, F-wave and V-wave) or mechanical stimulation of the tendon (T-reflex). In what follows we will discuss the methodology of these techniques as well as their modulation in response to a variety of conditionings.

## **4.1. The H-reflex**

The H-reflex was first described in 1918 by Paul Hoffmann [17] and is the electrical ho‐ mologous of the stretch reflex. It is elicited by a transcutaneous electrical stimulation (rectangular pulse with 1ms duration) applied to a mixed nerve that synchronously acti‐ vates afferent fibers from muscle spindles (see the arrow showing the orthodromic senso‐ ry activation in Figure 6). The evoked afferent volley generates excitatory post-synaptic potentials (EPSPs) in α-MNs (hereafter referred to as MNs) that may fire action poten‐ tials if they surpass the firing threshold. These EPSPs seem to be generated mainly by the monosynaptic Ia-MN excitatory pathway but they are also influenced by oligosynap‐ tic pathways [18]. The action potentials originating from the MNs lead to the generation of a CMAP recorded with surface EMG electrodes at the homonymous muscle. The evoked CMAP is termed H-reflex and is different from the interference pattern of EMG described in the preceding text (see sections 2 and 3), which is characterized by the asyn‐ chronous firing of MUs. The technique of H-reflex has been widely used to assess the ex‐ citability of the stretch reflex pathway and infer spinal cord mechanisms contributing towards motor control [1, 19]. In the lower limbs, the SO muscle has often been used be‐ cause its electrically-elicited reflex response is relatively easy to obtain.The muscle affer‐ ents of group I (Ia and Ib) and II are also depicted in the schematic of Figure 6. However, for low intensity stimulation, group I muscle afferents (mainly Ia) are primari‐ ly activated [20].

associated with the effect of the RI. To allow comparisons between subjects one may adopt the

Besides changing the excitability of MNs, pathways converging to the spinal cord may also affect the excitability of several spinal cord elements by acting on presynaptic terminals (e.g.,

**4. Conditioning of the evoked phasic ("synchronous") muscle activity**

So far, we have discussed the case of asynchronous voluntary activity of MUs that generates the EMG interference pattern. Another way to assess spinal cord processes is by means of

A variety of reflexes (stretch reflex, cutaneous reflex, H-reflex, etc) has been studied at rest, during locomotion and during the performance of a number of motor tasks in an attempt to better understand how the central nervous system (CNS) integrates the descending signals with those coming from the periphery [1]. Ascending signals from the periphery are incorpo‐ rated into motor plans in order to continuously update the CNS and generate suitable commands to muscles that will work in concert to produce a functionally relevant motor output. At the spinal cord level, the afferent influx coming from muscles, joints and skin help to sculpt motor behavior by playing a significant role in the modulation of the excitability of

The stretch reflex pathway is one of special interest and will be the focus of this topic. The excitability of this pathway (or parts of it) can be assessed by means of either electrical stimulation of peripheral nerve (H-reflex, F-wave and V-wave) or mechanical stimulation of the tendon (T-reflex). In what follows we will discuss the methodology of these techniques as

The H-reflex was first described in 1918 by Paul Hoffmann [17] and is the electrical ho‐ mologous of the stretch reflex. It is elicited by a transcutaneous electrical stimulation (rectangular pulse with 1ms duration) applied to a mixed nerve that synchronously acti‐ vates afferent fibers from muscle spindles (see the arrow showing the orthodromic senso‐ ry activation in Figure 6). The evoked afferent volley generates excitatory post-synaptic potentials (EPSPs) in α-MNs (hereafter referred to as MNs) that may fire action poten‐ tials if they surpass the firing threshold. These EPSPs seem to be generated mainly by the monosynaptic Ia-MN excitatory pathway but they are also influenced by oligosynap‐ tic pathways [18]. The action potentials originating from the MNs lead to the generation of a CMAP recorded with surface EMG electrodes at the homonymous muscle. The evoked CMAP is termed H-reflex and is different from the interference pattern of EMG described in the preceding text (see sections 2 and 3), which is characterized by the asyn‐ chronous firing of MUs. The technique of H-reflex has been widely used to assess the ex‐

ratio of the latter to the former as an index of the level of RI.

64 Electrodiagnosis in New Frontiers of Clinical Research

the Ia-MN synapse). Presynaptic effects will be discussed later.

reflex-generated compound muscle action potentials (CMAP).

well as their modulation in response to a variety of conditionings.

different reflex pathways [1].

**4.1. The H-reflex**

The presence of a stable M-wave (direct motor response, see below) is desired in most studies to assure (by indirect means) a constant stimulation (see the arrow showing the orthodromic motor activation in Figure 6). Thus, any changes in H-reflex amplitude would be related to neurophysiological factors and not to alterations in stimulus efficacy, which would change the M-wave as well [21].

**Figure 6.** Schematic of the stretch reflex pathway and the mixed nerve stimulation that generates orthodromic and antidromic nerve activity (only the monosynaptic pathway from the Ia is shown). EMG trace showing an H-reflex and M-wave elicited by a transcutaneous electrical stimulus (1ms duration) applied through electrodes located over the skin at the popliteal fossa. The stimulus activates Ia afferent and motor axons from the mixed nerve (PTN) that supplies the SO muscle. The resulting H-reflex and M-wave are recorded with surface electrodes (see the schematic on the left). **A**: Stimulus artifact indicating when the stimulus was delivered; **M**: M-wave; **H**: H-reflex.

#### *4.1.1. Recruitment order of reflexively activated motoneurons*

With the increase of the stimulus intensity, a larger number of Ia afferents are activated leading to reflex recruitment of more MNs. The MNs in the spinal cord are synaptically recruited according to the size principle [22], i.e., the small size MUs (with low threshold for synaptic input) are recruited first. Therefore, H-reflexes of low amplitude reflect the activation of small MUs (see Figure 7). Higher amplitudes of H-reflex correspond to the activation of intermedi‐ ated sized MUs along with the small ones. The increment in H-reflex amplitude reaches a limit that is not only related to the number of MNs within the pool, but also to the phenomenon of "annihilation", i.e., action potentials in the efferent axon generated reflexively collide with the antidromic volley due to the firing of the distal part of the efferent axon by the electrical stimulus (Figure 8). Therefore, those motor axons that were activated by the transcutaneous electrical stimulation generate antidromic spikes (shown in Figure 6 as the "antidromic motor activation") that prevent the action potentials of reflex origin from reaching the muscle (see Figure 8). As the axonal conduction velocity of efferents is lower than the afferents, there is enough time for the collision to take place in the efferent axons. The action potentials generated in the efferent axons also propagate toward the muscle (orthodromic motor activation in Figure 6 and red arrows in Figure 8) and will generate a shorter latency response (M-wave). The stimulus intensity that generates the lowest amplitude M-wave is termed motor threshold (MT). The direct motor response (M-wave) increases monotonically with stimulus intensity until its maximum (MMAX), as there is no annihilation in the distal part of the motor axons (distal to the stimulation point; see Figure 8). A supramaximal stimulus intensity will discharge 100% of the efferent axons, yielding the MMAX and blocking the generation of any H-reflex response due to the antidromic motor volley (see Figure 7).

**Figure 8.** Schematics illustrating the recruitment of sensory and motor fibers by transcutaneous electrical stimulus. The stimulus intensity increases from **a** to **d** (see also the corresponding A-D regions in Figure 7) but the duration is always the same, 1ms. Note the bidirectional propagation of potentials (toward the spinal cord and muscle) in **b**-**d**. The blue arrows indicate the afferent volley (travelling across the blue axons) and the action potentials reflexively evoked in the motor axons (green cells) that will generate the H-reflex. The red arrows represent the orthodromic mo‐ tor activation (see also Figure 6) that will generate the M-wave. The green arrows represent the antidromic volleys in either sensory or motor axons. The green arrows in the motor axons will cause a collision (indicated by a yellow star) with the reflexively evoked volley (blue arrows) in the efferent fibers. **a)** For low intensity stimulus the smallest MUs (filled circles) are recruited according to the size principle and no collision is observed. At this point only the H-reflex (without M-wave) is present in the EMG recording (see also Figure 7). **b)** With the increased intensity of electrical stim‐ ulation (1ms rectangular pulse), a few motor axons discharge action potentials that propagate antidromically leading to the annihilation of spikes. At this point the H-reflex is accompanied by an M-wave in the EMG recording. The Mwave has a shorter latency than the H-reflex because it is a direct response, i.e., it does not travel to the spinal cord and back to the muscle (red arrows reach the muscle before the blue ones). **c)** The stimulus intensity is much higher and the collision occurs in a larger number of efferent axons, despite the number of afferents recruited by the electrical stimulation being the same (or even higher) than in situation **b**. At this point the H-reflex amplitude is lower than the M-wave (C region of Figure 7b). **d)** The supramaximal intensity recruits 100% of the sensory and motor fibers inducing 100% of annihilation. No H-reflex is identified in the EMG recording and the M-wave reaches its maximal amplitude

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In this scenario, the H-reflex will never reflect the activation of all the MNs within the pool, even if the stimulus intensity is increased. Instead, this reflex response reaches a maximum (HMAX) as a result of a balance between mechanisms that tend to change the reflex amplitude

(MMAX) (D region of Figure 7b).

**Figure 7. a)** EMG traces recorded from the SO muscle showing changes in H-reflex and M-wave amplitudes as a func‐ tion of stimulus intensity (unpublished data). Note the extinction of the H-reflex when the MMAX is present in the re‐ cording (under the maximal stimulus intensity). **b)** Schematic recruitment curve with the peak-to-peak values of Hreflexes (blue) and M-waves (red) along the stimulus intensity. The regions A-D delimited by the green dashed vertical lines contain, respectively: the ascending limb of recruitment curve; motor threshold and HMAX; descending limb of the curve; MMAX. **c)** Recruitment curve obtained from the SO muscle of one representative subject. Data based on [10]. The circles and crosses represent the peak-to-peak amplitude values of the H-reflex and M-wave, respectively. **d)** Same da‐ ta from **c** showing a sigmoidal fit to the ascending limb of the H-reflex recruitment curve. Data based on [9], but fig‐ ures are unpublished.

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antidromic volley due to the firing of the distal part of the efferent axon by the electrical stimulus (Figure 8). Therefore, those motor axons that were activated by the transcutaneous electrical stimulation generate antidromic spikes (shown in Figure 6 as the "antidromic motor activation") that prevent the action potentials of reflex origin from reaching the muscle (see Figure 8). As the axonal conduction velocity of efferents is lower than the afferents, there is enough time for the collision to take place in the efferent axons. The action potentials generated in the efferent axons also propagate toward the muscle (orthodromic motor activation in Figure 6 and red arrows in Figure 8) and will generate a shorter latency response (M-wave). The stimulus intensity that generates the lowest amplitude M-wave is termed motor threshold (MT). The direct motor response (M-wave) increases monotonically with stimulus intensity until its maximum (MMAX), as there is no annihilation in the distal part of the motor axons (distal to the stimulation point; see Figure 8). A supramaximal stimulus intensity will discharge 100% of the efferent axons, yielding the MMAX and blocking the generation of any H-reflex

**Figure 7. a)** EMG traces recorded from the SO muscle showing changes in H-reflex and M-wave amplitudes as a func‐ tion of stimulus intensity (unpublished data). Note the extinction of the H-reflex when the MMAX is present in the re‐ cording (under the maximal stimulus intensity). **b)** Schematic recruitment curve with the peak-to-peak values of Hreflexes (blue) and M-waves (red) along the stimulus intensity. The regions A-D delimited by the green dashed vertical lines contain, respectively: the ascending limb of recruitment curve; motor threshold and HMAX; descending limb of the curve; MMAX. **c)** Recruitment curve obtained from the SO muscle of one representative subject. Data based on [10]. The circles and crosses represent the peak-to-peak amplitude values of the H-reflex and M-wave, respectively. **d)** Same da‐ ta from **c** showing a sigmoidal fit to the ascending limb of the H-reflex recruitment curve. Data based on [9], but fig‐

response due to the antidromic motor volley (see Figure 7).

66 Electrodiagnosis in New Frontiers of Clinical Research

ures are unpublished.

**Figure 8.** Schematics illustrating the recruitment of sensory and motor fibers by transcutaneous electrical stimulus. The stimulus intensity increases from **a** to **d** (see also the corresponding A-D regions in Figure 7) but the duration is always the same, 1ms. Note the bidirectional propagation of potentials (toward the spinal cord and muscle) in **b**-**d**. The blue arrows indicate the afferent volley (travelling across the blue axons) and the action potentials reflexively evoked in the motor axons (green cells) that will generate the H-reflex. The red arrows represent the orthodromic mo‐ tor activation (see also Figure 6) that will generate the M-wave. The green arrows represent the antidromic volleys in either sensory or motor axons. The green arrows in the motor axons will cause a collision (indicated by a yellow star) with the reflexively evoked volley (blue arrows) in the efferent fibers. **a)** For low intensity stimulus the smallest MUs (filled circles) are recruited according to the size principle and no collision is observed. At this point only the H-reflex (without M-wave) is present in the EMG recording (see also Figure 7). **b)** With the increased intensity of electrical stim‐ ulation (1ms rectangular pulse), a few motor axons discharge action potentials that propagate antidromically leading to the annihilation of spikes. At this point the H-reflex is accompanied by an M-wave in the EMG recording. The Mwave has a shorter latency than the H-reflex because it is a direct response, i.e., it does not travel to the spinal cord and back to the muscle (red arrows reach the muscle before the blue ones). **c)** The stimulus intensity is much higher and the collision occurs in a larger number of efferent axons, despite the number of afferents recruited by the electrical stimulation being the same (or even higher) than in situation **b**. At this point the H-reflex amplitude is lower than the M-wave (C region of Figure 7b). **d)** The supramaximal intensity recruits 100% of the sensory and motor fibers inducing 100% of annihilation. No H-reflex is identified in the EMG recording and the M-wave reaches its maximal amplitude (MMAX) (D region of Figure 7b).

In this scenario, the H-reflex will never reflect the activation of all the MNs within the pool, even if the stimulus intensity is increased. Instead, this reflex response reaches a maximum (HMAX) as a result of a balance between mechanisms that tend to change the reflex amplitude when the stimulus is increased. The main mechanism that increases H-reflex amplitude (assuming the subject is in a relaxed and controlled state) is the larger number of Ia axons activated by the higher intensity stimulus. The main mechanism that decreases the H-reflex amplitude in response to a stimulus intensity increase is the above mentioned collision of action potentials in the efferent axons. Other mechanisms that may also contribute to decrease the H-reflex amplitude for a higher stimulus amplitude include (1) the activation of Ib afferents (see schematic in Figure 6) [20], (2) the activation of large-diameter cutaneous afferents, (3) the firing of Renshaw cells in response to the synchronous antidromic (or orthodromic) firing of MNs in the pool [19, 23]. These longer latency mechanisms have their putative effect on Hreflex amplitude because the later phases of the H-reflex waveform (after its rise) have been associated with the longer latency oligosynaptic pathways that excite the MNs [18]. For stimulus intensities above that corresponding to HMAX (from the beginning of the descending phase of the recruitment curve, Figure 7b and c), the larger the number of efferents undergoing collision the lower the amplitude of the H-reflex (see Figure 7 and Figure 8).

time, i.e., when the refractory period ceases. This generates a rebound effect that can be seen in the EMG recordings of Figure 10. Other mechanisms might be involved in the generation of the silent period such as recurrent inhibition from Renshaw cells [27, 28]. This silent period has been shown to be useful, e.g. for the quantification of the degree of crosstalk between two muscles [14] and for the study of involuntary sustained muscle contraction after a train of

**Figure 9.** EMG recordings from the SO muscle showing the H-reflexes elicited in two different conditions, at rest and during isometric voluntary contraction (unpublished data). The black trace represents the averaged response.

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S R


**Figure 10.** EMG recordings (unpublished data; *n* = 50) from the SO muscle during upright stance showing 100ms of background muscle activity (B) prior to the delivery of a stimulus to the PTN (A) to generate an H-reflex (H). Following the H-reflex, a clear silent period (S) of ~100ms is noticed. A rebound effect (R) can also be seen in the interval be‐

Not only tonic voluntary contraction induces changes in reflex excitability. There are pre- and post-synaptic influences that affect H-reflex amplitude from a variety of sources. Presynaptic inhibition (PSI) is perhaps one of the most important mechanisms of reflex modulation [30]. By means of PSI the CNS can regulate the excitability of the stretch reflex pathway in different

stimuli [29].

tween 150 and 200ms.

B

A

H

It is always recommended keeping the amplitude of the test H-reflex in the ascending limb of the recruitment curve, where there is no (or very few) collision in the motor axons. The best fit for the ascending limb of the curve is a sigmoid (Figure 7d) [24]. This fitting is important to define some parameters that can be extracted from the curve, such as slope, current threshold and HMAX [10] (see ahead). It is also highly recommended using a test H-reflex amplitude within the range of 20-30%MMAX [25] because at this amplitude reflexes are more responsive to conditioning.

## *4.1.2. H-reflex amplitude and ongoing EMG activity under different conditions*

The H-reflex can be evoked in different conditions: at rest, during voluntary muscle contrac‐ tion, in upright stance, during rhythmic movements of different limbs, during walking, running, and so on [1, 10, 26]. Usually, H-reflex evoked during contraction of the homonymous muscle shows higher amplitude compared to H-reflex evoked at rest [1, 21] (Figure 9). This happens because the MNs that were not fired by the afferent volley caused by the electrical stimulus might reach the firing threshold during contraction due to the summation of EPSPs generated by the activation of DTs. Figure 9 shows an example of H-reflex obtained at rest and during tonic voluntary isometric contraction of the SO muscle. When the level of voluntary contraction increases (more MUs are recruited), the size of the H-reflex increases in parallel [26]. Therefore, care should be taken when the objective is to study the modulation of the Hreflex during motor activity as its amplitude depends on the excitability of the MNs in the pool [21]. In practical terms, it is crucial to maintain a constant level of muscle activity throughout the experiment [1, 21].

During a sustained voluntary contraction there is a momentary silence in the muscle activity (silent period) following the H-reflex, as seen in the EMG recording of Figure 10. The silent period is mainly ascribed to the after-hyperpolarization (AHP) of the MNs after the synchro‐ nous reflex activation, since the EPSPs caused by descending commands cannot ellicit another spike in the MN during its refractory period (which is related to the AHP). After this period, the constant descending drive causes the MNs to reach the firing threshold almost at the same Experimental and Simulated EMG Responses in the Study of the Human Spinal Cord http://dx.doi.org/10.5772/54870 69

when the stimulus is increased. The main mechanism that increases H-reflex amplitude (assuming the subject is in a relaxed and controlled state) is the larger number of Ia axons activated by the higher intensity stimulus. The main mechanism that decreases the H-reflex amplitude in response to a stimulus intensity increase is the above mentioned collision of action potentials in the efferent axons. Other mechanisms that may also contribute to decrease the H-reflex amplitude for a higher stimulus amplitude include (1) the activation of Ib afferents (see schematic in Figure 6) [20], (2) the activation of large-diameter cutaneous afferents, (3) the firing of Renshaw cells in response to the synchronous antidromic (or orthodromic) firing of MNs in the pool [19, 23]. These longer latency mechanisms have their putative effect on Hreflex amplitude because the later phases of the H-reflex waveform (after its rise) have been associated with the longer latency oligosynaptic pathways that excite the MNs [18]. For stimulus intensities above that corresponding to HMAX (from the beginning of the descending phase of the recruitment curve, Figure 7b and c), the larger the number of efferents undergoing

It is always recommended keeping the amplitude of the test H-reflex in the ascending limb of the recruitment curve, where there is no (or very few) collision in the motor axons. The best fit for the ascending limb of the curve is a sigmoid (Figure 7d) [24]. This fitting is important to define some parameters that can be extracted from the curve, such as slope, current threshold and HMAX [10] (see ahead). It is also highly recommended using a test H-reflex amplitude within the range of 20-30%MMAX [25] because at this amplitude reflexes are more responsive to

The H-reflex can be evoked in different conditions: at rest, during voluntary muscle contrac‐ tion, in upright stance, during rhythmic movements of different limbs, during walking, running, and so on [1, 10, 26]. Usually, H-reflex evoked during contraction of the homonymous muscle shows higher amplitude compared to H-reflex evoked at rest [1, 21] (Figure 9). This happens because the MNs that were not fired by the afferent volley caused by the electrical stimulus might reach the firing threshold during contraction due to the summation of EPSPs generated by the activation of DTs. Figure 9 shows an example of H-reflex obtained at rest and during tonic voluntary isometric contraction of the SO muscle. When the level of voluntary contraction increases (more MUs are recruited), the size of the H-reflex increases in parallel [26]. Therefore, care should be taken when the objective is to study the modulation of the Hreflex during motor activity as its amplitude depends on the excitability of the MNs in the pool [21]. In practical terms, it is crucial to maintain a constant level of muscle activity throughout

During a sustained voluntary contraction there is a momentary silence in the muscle activity (silent period) following the H-reflex, as seen in the EMG recording of Figure 10. The silent period is mainly ascribed to the after-hyperpolarization (AHP) of the MNs after the synchro‐ nous reflex activation, since the EPSPs caused by descending commands cannot ellicit another spike in the MN during its refractory period (which is related to the AHP). After this period, the constant descending drive causes the MNs to reach the firing threshold almost at the same

collision the lower the amplitude of the H-reflex (see Figure 7 and Figure 8).

*4.1.2. H-reflex amplitude and ongoing EMG activity under different conditions*

conditioning.

68 Electrodiagnosis in New Frontiers of Clinical Research

the experiment [1, 21].

**Figure 9.** EMG recordings from the SO muscle showing the H-reflexes elicited in two different conditions, at rest and during isometric voluntary contraction (unpublished data). The black trace represents the averaged response.

time, i.e., when the refractory period ceases. This generates a rebound effect that can be seen in the EMG recordings of Figure 10. Other mechanisms might be involved in the generation of the silent period such as recurrent inhibition from Renshaw cells [27, 28]. This silent period has been shown to be useful, e.g. for the quantification of the degree of crosstalk between two muscles [14] and for the study of involuntary sustained muscle contraction after a train of stimuli [29].

**Figure 10.** EMG recordings (unpublished data; *n* = 50) from the SO muscle during upright stance showing 100ms of background muscle activity (B) prior to the delivery of a stimulus to the PTN (A) to generate an H-reflex (H). Following the H-reflex, a clear silent period (S) of ~100ms is noticed. A rebound effect (R) can also be seen in the interval be‐ tween 150 and 200ms.

Not only tonic voluntary contraction induces changes in reflex excitability. There are pre- and post-synaptic influences that affect H-reflex amplitude from a variety of sources. Presynaptic inhibition (PSI) is perhaps one of the most important mechanisms of reflex modulation [30]. By means of PSI the CNS can regulate the excitability of the stretch reflex pathway in different motor contexts. For instance, it is generally accepted that PSI increases from the standing position to walking and even more during running [26].

Even in motor tasks involving rhythmic movements of limbs that mimic patterns of locomotor movements (e.g., arm swing during walking) modulation of reflex responses can be observed. It has been suggested that arm cycling in an ergometer decreases reflex amplitude of the SO muscle by increasing the level of PSI [31]. This result has been used as an evidence for the exis‐ tence of a neuronal linkage between upper and lower limbs responsible for coordinated actions during locomotion [10]. An example of reduced amplitude H-reflex is shown in Figure 11.

**Figure 11.** Comparison of H-reflex amplitude from the SO muscle at rest and during arm cycling. The constant amplit tude of the M-wave indicates that there were no changes in stimulus efficacy. The black trace represents the averaged response. Data based on [32], but figures are unpublished.

stimulus will have a lower amplitude as compared to the H-reflex elicited without conditioning due to the PSI effect. This procedure has been widely used in many research laboratories to

**Figure 12.** Recruitment curves obtained in two distinct conditions, at rest (blue) and during rhythmic arm movement (red). **a)** It is possible to note a decrease in HMAX amplitude along with a right shift of the red curve. Note that the Mwave recruitment was very similar in both conditions. **b)** A closer inspection reveals a slight change in recruitment gain as indicated by the steeper slope of the blue curve compared to the red one. A clear change in H-reflex threshold can

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Another pre-synaptic mechanism that affects H-reflex amplitude is post-activation depression (or homosynaptic depression - HD), which consists in a frequency-dependent reduction of reflex amplitude, i.e., when the stimuli are applied with frequencies higher than 0.1Hz (less than 10s interval) a depression in H-reflex amplitude is observed supposedly due to a reduced release of neurotransmitter in the Ia terminal [37, 38]. The HD is also exemplified in the upper panel of Figure 13b (green curve) that shows an averaged reflex response evoked at every 1s

It is interesting to note a further decrease in H-reflex amplitude when both presynaptic mechanisms are present (PSI+HD; see the blue trace in Figure 13b). This result might be related to the increased frequency used for the conditioning stimulus (1Hz as compared to 0.1Hz used to obtain the trace in green) delivered 100ms before the test stimulus (also delivered at 1Hz to induce HD). Indeed, it was recently shown that an increased conditioning stimulus frequency

In section 4.1 we presented a technique for the assessment of stretch reflex excitability based on transcutaneous electrical stimulation (the H-reflex). Here we are going to discuss another way to investigate the same pathway by using a mechanical stimulus applied to the tendon in opposition to the electrical current applied to a peripheral mixed nerve. The target again will be the SO muscle. This technique has been used by clinicians to assess the integrity of the spinal

investigate changes in the degree of PSI in different conditions.

also be observed. Data based on [10], but figures are unpublished.

(1Hz stimulus frequency).

**4.2. The T-reflex**

enhances PSI of both H- [39] and T-reflexes [36].

It is also possible to explore a wider range of MUs by examining the behavior of the H-reflex evoked at different stimulus intensities during the performance of a motor task. Therefore, instead of comparing test reflex responses of a given amplitude (just like those shown in Figure 11) that would represent a single point in the recruitment curve (hence, a limited fraction of active MUs within the pool), the whole recruitment curve can be analyzed (Figure 12). Several parameters may then be extracted from the recruitment curve and compared across conditions [10, 24] and the input-output relations of the system under study can be properly examined. Figure 12 shows an example of changes in the SO recruitment curve during rhythmic arm movements using a stepping ergometer. One can notice a reduction in HMAX values as well as a right shifting of the curve, indicating changes in the threshold of reflex response (see the right panel of Figure 12). It is also possible to investigate changes in the recruitment gain by comparing the slope of the ascending curve between conditions. Note that no significant changes occurred in the M-wave curve (crosses), indicating that the stimulus efficacy was constant for both conditions.

In an attempt to better describe mechanisms responsible for reflex modulation, protocols based on conditioning stimulation have been developed. For example, it is possible to assess the level of PSI under different conditions [33, 34]. The technique (illustrated in Figure 13a) consists in applying a conditioning electrical stimulus to the CPN (1ms rectangular pulse) and a test stimulus to the PTN with a conditioning-to-test (CT) interval of 100ms [35] (compare gray and red traces in the upper panel of Figure 13b). The reflex response conditioned by the CPN Experimental and Simulated EMG Responses in the Study of the Human Spinal Cord http://dx.doi.org/10.5772/54870 71

**Figure 12.** Recruitment curves obtained in two distinct conditions, at rest (blue) and during rhythmic arm movement (red). **a)** It is possible to note a decrease in HMAX amplitude along with a right shift of the red curve. Note that the Mwave recruitment was very similar in both conditions. **b)** A closer inspection reveals a slight change in recruitment gain as indicated by the steeper slope of the blue curve compared to the red one. A clear change in H-reflex threshold can also be observed. Data based on [10], but figures are unpublished.

stimulus will have a lower amplitude as compared to the H-reflex elicited without conditioning due to the PSI effect. This procedure has been widely used in many research laboratories to investigate changes in the degree of PSI in different conditions.

Another pre-synaptic mechanism that affects H-reflex amplitude is post-activation depression (or homosynaptic depression - HD), which consists in a frequency-dependent reduction of reflex amplitude, i.e., when the stimuli are applied with frequencies higher than 0.1Hz (less than 10s interval) a depression in H-reflex amplitude is observed supposedly due to a reduced release of neurotransmitter in the Ia terminal [37, 38]. The HD is also exemplified in the upper panel of Figure 13b (green curve) that shows an averaged reflex response evoked at every 1s (1Hz stimulus frequency).

It is interesting to note a further decrease in H-reflex amplitude when both presynaptic mechanisms are present (PSI+HD; see the blue trace in Figure 13b). This result might be related to the increased frequency used for the conditioning stimulus (1Hz as compared to 0.1Hz used to obtain the trace in green) delivered 100ms before the test stimulus (also delivered at 1Hz to induce HD). Indeed, it was recently shown that an increased conditioning stimulus frequency enhances PSI of both H- [39] and T-reflexes [36].

#### **4.2. The T-reflex**

motor contexts. For instance, it is generally accepted that PSI increases from the standing

Even in motor tasks involving rhythmic movements of limbs that mimic patterns of locomotor movements (e.g., arm swing during walking) modulation of reflex responses can be observed. It has been suggested that arm cycling in an ergometer decreases reflex amplitude of the SO muscle by increasing the level of PSI [31]. This result has been used as an evidence for the exis‐ tence of a neuronal linkage between upper and lower limbs responsible for coordinated actions during locomotion [10]. An example of reduced amplitude H-reflex is shown in Figure 11.

**Figure 11.** Comparison of H-reflex amplitude from the SO muscle at rest and during arm cycling. The constant amplit tude of the M-wave indicates that there were no changes in stimulus efficacy. The black trace represents the averaged

It is also possible to explore a wider range of MUs by examining the behavior of the H-reflex evoked at different stimulus intensities during the performance of a motor task. Therefore, instead of comparing test reflex responses of a given amplitude (just like those shown in Figure 11) that would represent a single point in the recruitment curve (hence, a limited fraction of active MUs within the pool), the whole recruitment curve can be analyzed (Figure 12). Several parameters may then be extracted from the recruitment curve and compared across conditions [10, 24] and the input-output relations of the system under study can be properly examined. Figure 12 shows an example of changes in the SO recruitment curve during rhythmic arm movements using a stepping ergometer. One can notice a reduction in HMAX values as well as a right shifting of the curve, indicating changes in the threshold of reflex response (see the right panel of Figure 12). It is also possible to investigate changes in the recruitment gain by comparing the slope of the ascending curve between conditions. Note that no significant changes occurred in the M-wave curve (crosses), indicating that the stimulus efficacy was

In an attempt to better describe mechanisms responsible for reflex modulation, protocols based on conditioning stimulation have been developed. For example, it is possible to assess the level of PSI under different conditions [33, 34]. The technique (illustrated in Figure 13a) consists in applying a conditioning electrical stimulus to the CPN (1ms rectangular pulse) and a test stimulus to the PTN with a conditioning-to-test (CT) interval of 100ms [35] (compare gray and red traces in the upper panel of Figure 13b). The reflex response conditioned by the CPN

position to walking and even more during running [26].

70 Electrodiagnosis in New Frontiers of Clinical Research

response. Data based on [32], but figures are unpublished.

constant for both conditions.

In section 4.1 we presented a technique for the assessment of stretch reflex excitability based on transcutaneous electrical stimulation (the H-reflex). Here we are going to discuss another way to investigate the same pathway by using a mechanical stimulus applied to the tendon in opposition to the electrical current applied to a peripheral mixed nerve. The target again will be the SO muscle. This technique has been used by clinicians to assess the integrity of the spinal cord after injury or in neuropathologies [40]. Perhaps, the main concern about the use of this technique in scientific research is to maintain the mechanical stimulus consistent throughout the experiment. Several investigators have used an instrumented hammer [41, 42] designed to apply a somewhat controlled mechanical percussion to the tendon. An alternative approach is to use a powerful electromechanical shaker to achieve tendon mechanical stimulation [43]. The tip of the shaker is lightly pressed against the Achilles tendon to ensure reasonable stimulus reproducibility. The shaker can be controlled via software that provides the desired input waveform shape, amplitude and duration (e.g., a sinusoidal cycle with 10ms duration and excursion of ~3mm). An inbuilt accelerometer is a reliable alternative to provide a feedback from the shaker tip excursion and monitoring stimulus consistency [3, 43].

interest. The mechanical percussion induces a brief muscle stretch leading to the activation of spindle afferents. As a consequence, the mechanical stimulus generates a burst of firing in each afferent axon (mainly in Ia afferents). In contrast, the electrical stimulation produces only one spike per axon and at a more fixed latency (less sparse spikes arriving to the MN pool) than the burst due to the tendon tap [20] (see dashed circle in Figure 13). Therefore, the effect of asynchronous afferent bursts on the Ia-MN synapses will be different from a less dispersed volley of single action potentials. The MN depolarization (sum of EPSPs) generated by a more asynchronous afferent volley would produce a long rising time course in the membrane of the MN, giving time to other inputs (e.g., Ib afferents; see also Figure 6) mediated by oligosynaptic pathways to exert influence on the membrane of the postsynaptic cell [20]. Therefore, condi‐ tioning effects on T-reflex might be different from effects observed on H-reflex. For instance, T-reflex has been shown to be less responsive to a conditioning that induces PSI compared to the H-reflex [44] (see Figure 13). Despite the relatively lower sensitivity to PSI, the T-reflex also showed a stronger inhibitory effect when the conditioning stimulus was applied at higher frequency (1Hz), as for the H-reflex (see previous section) [36]. However, postsynaptic effects (e.g., mediated by RI) may have similar strength for both reflexes (see section 5.2.1; [44, 45])

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Another important difference is related to the sensitivity of reflex responses to the fusimotor system excitability. T-reflexes are differentially susceptible to γ-MN activity (that regulates the muscle spindle sensitivity) as compared to H-reflexes [46]. All these aspects need to be taken into account in the interpretation of results and/or comparisons between both types of reflex

F-waves are recorded routinely in clinical neurophysiological practice [47]. The F wave is a late response that occurs in a muscle following stimulation of its motor nerve, evoked by antidromic activation ("backfiring") of a fraction of the MNs. Typically, F-waves are evoked in response to a strong electrical stimulus (supramaximal stimulation) applied to a peripheral nerve. Action potentials traveling orthodromically reach the muscle fiber, thereby eliciting a strong M-response (MMAX). The action potentials traveling antidromically (see arrows in Figure 6) reach the cell bodies of the MNs making a small fraction of them to fire. This causes orthodromic action potentials to travel back towards the muscle, generating a relatively small amplitude CMAP called F-wave. Several measurements can be done on the F responses, including peak-to-peak amplitude, duration, latency (period between stimulation and F-wave response), and persistency (number of F-waves obtained per number of stimulations). Most electrophysiologists agree that F-wave latency constitutes a valuable parameter that reflects conduction properties of motor axons, being even more reliable than distal motor conduction measurements used to detect mild or early generalized abnormalities [48]. Although the use of F waves for assessing MN excitability is controversial [49], they are sensitive to changes in MN excitability [48] and have been used to assess it in a variety of protocols [50, 51]. In contrast to the H-reflex, which is influenced by presynaptic effects (PSI and HD), the F response is not a reflex (is not elicited by Ia volley), hence its generation is related solely to the MN membrane

regardless of the stimulus frequency.

responses.

**4.3. The F-wave**

**Figure 13. a)** Schematic of the experimental setup for testing the presynaptic inhibition pathway. The test stimulus was either electrical (Stim2) or mechanical (indicated by the shaker in contact with the SO tendon). The interval be‐ tween conditioning (Stim 1) and test stimuli (CT interval) was 100ms. SO afferent activation is shown in the dashed circle. For the H-reflex a single action potential is generated per Ia fiber, whereas the tendon stimulus evokes a burst of firing in Ia afferents. This difference might be responsible for the lesser sensitivity of the T-reflex to PSI as compared to the H-reflex (see text for details). **b)** Upper panel: Averaged H-reflex waveforms obtained in the SO muscle under different conditionings as compared to the control or no-conditioning case (in dark gray). The trace in red shows an Hreflex conditioned by a 1ms stimulus to the CPN to induce PSI on the afferent terminals of the SO muscle. The green trace represents the H-reflex under homosynaptic depression, HD (stimulus applied to the PTN at 1Hz). When the test and conditioned responses were obtained with interval of 1s (conditioned and test stimuli applied at 1Hz) an addi‐ tional inhibition was observed (blue trace; HD+PSI). The vertical arrow shows the instant of stimulus delivery. Lower panel: the same as for upper panel showing the T-reflex. Note the longer delay as compared to the H-reflex (indicated by a horizontal dashed line). Results similar to those observed in H-reflex were attained for condition PSI+HD. Data based on [36], but figures are unpublished.

The main difference between both techniques (H and T reflexes) is that in the case of the Hreflex the stimulus bypasses the muscle spindles (it is applied directly to the nerve, see Figure 6). To generate the T-reflex the stimulus is applied distally, on the tendon of the muscle of interest. The mechanical percussion induces a brief muscle stretch leading to the activation of spindle afferents. As a consequence, the mechanical stimulus generates a burst of firing in each afferent axon (mainly in Ia afferents). In contrast, the electrical stimulation produces only one spike per axon and at a more fixed latency (less sparse spikes arriving to the MN pool) than the burst due to the tendon tap [20] (see dashed circle in Figure 13). Therefore, the effect of asynchronous afferent bursts on the Ia-MN synapses will be different from a less dispersed volley of single action potentials. The MN depolarization (sum of EPSPs) generated by a more asynchronous afferent volley would produce a long rising time course in the membrane of the MN, giving time to other inputs (e.g., Ib afferents; see also Figure 6) mediated by oligosynaptic pathways to exert influence on the membrane of the postsynaptic cell [20]. Therefore, condi‐ tioning effects on T-reflex might be different from effects observed on H-reflex. For instance, T-reflex has been shown to be less responsive to a conditioning that induces PSI compared to the H-reflex [44] (see Figure 13). Despite the relatively lower sensitivity to PSI, the T-reflex also showed a stronger inhibitory effect when the conditioning stimulus was applied at higher frequency (1Hz), as for the H-reflex (see previous section) [36]. However, postsynaptic effects (e.g., mediated by RI) may have similar strength for both reflexes (see section 5.2.1; [44, 45]) regardless of the stimulus frequency.

Another important difference is related to the sensitivity of reflex responses to the fusimotor system excitability. T-reflexes are differentially susceptible to γ-MN activity (that regulates the muscle spindle sensitivity) as compared to H-reflexes [46]. All these aspects need to be taken into account in the interpretation of results and/or comparisons between both types of reflex responses.
