Characteristic features of doublet-firing MUs are collected in **Table 1**. MUs were classified into three groups. MUs from group **S** fired occasional *single doublets* interspersed in regular discharge (see the example in **Figure 1A**); these doublets constituted less than 0.5 % of all discharges. MUs from the group **R** fired both *single* and *repetitive doublets*, i.e., the series of doublet and post-doublet discharges; in this group, the doublets constituted more than 6% of all discharges (see the example in **Figure 1B**). Group **E** comprised two MUs from one subject, firing *exceptional doublets* (about 1% of all discharges). This group will be described separately with more detail.

In **Figure 2** interval distributions of the two MUs with doublets are shown. Both have two separate maxima, one for regular discharges and the other for doublets. The histogram in **Figure 2A** has an additional maximum at around 36 ms, formed by six extremely long triplet intervals (35.9–36.5 ms; for more detailed description see the following section). **Figure 2B** presents the histogram of the MU with exceptional doublets. The duration of intradoublet ISI for this MU is within the limits 35.8–37.0 ms. Note striking similarity between both interval ranges (cf. also **Figure 5**).

**FIGURE 2 | Interval histograms of doublet-firing MUs. (A)** MU from group R. **(B)** MU from group E (see text for the explanation of the acronyms); 1, single, 2, double, 3, triple discharges. Note the coincidence of timing of triplets in A with doublets in B and some outsiders in both distributions.

In **Figure 3** the joint intradoublet ISI histogram for SOL MUs is presented with expanded scale. Exceptional doublets **(E)** create here a narrow maximum at about 37 ms, which differs considerably from the broader maximum of usual doublets **(U)**.

#### **UNUSUAL MULTIPLE DISCHARGES**

As already mentioned above, in two MUs of one subject exceptional doublets were observed, whose intradoublet ISI considerably exceeded the usual limits of 2.5–20 ms (set by the standards of electrophysiological terminology (AAEE, 1987; AAEM, 2001). **Figure 4** illustrates a long section of the discharge of a MU with exceptional doublets. The unit was discharging

#### **Table 1 | Firing characteristics of MU groups**.

*\* Calculated with respect to total number of MU spikes.*

*\*\* Interval between second and third discharge.*

around 6.5 Hz and occasionally slowed down below 5 Hz. It began to fire doublets about 8 min after the start of the experiment and continued until its end.

The intervals of these doublets exhibited much less variability (coefficient of variation 1.04%) than those of the usual doublets (12.0% for repetitive and 21.4% for single doublets). They were also accompanied by the prolonged post-doublet ISI.

The special class of unusual multiple discharges are triplets, which are much more seldom than doublets (cf. **Table 1**). In our experimental data collected from SOL, we encountered only one MU firing triplets. Triplets presented the stereotyped firing pattern (**Figure 5**): the interval between second and third discharge (triplet ISI) was substantially longer than that between first and second discharge (intradoublet ISI).

Surprisingly, the triplet ISI duration was virtually equal that of the exceptional intradoublet ISI (**Figure 5**, cf. also **Figure 2** and **Table 1**). This coincidence is quite remarkable given that the exceptional doublets and the triplets were recorded from two different subjects.

#### **DISCUSSION**

This paper presents the doublets recorded from SOL muscle, which were found unexpectedly among single MU data collected for other purposes. SOL is the muscle perhaps most frequently investigated in human studies (e.g., Person and Kudina, 1972; Ashby and Labelle, 1977; Sabbahi and Sedgwick, 1987; Kudina, 1988; Kudina and Pantseva, 1988; Miles et al., 1989; Türker and Miles, 1991; Person and Kozhina, 1992; Kiehn and Eken, 1997; Türker et al., 1997). However, doublets have never been reported in this muscle. Even in the experiments testing the excitability of human MUs within the ISI none of the 141 SOL MUs was found to reveal any sign of increased excitability in the initial interval fragment (Sabbahi and Sedgwick, 1987; Kudina, 1988) in contrast to MUs from flexor carpi ulnaris capable of firing doublets (Kudina and Churikova, 1990). Thus, the occurrence of doublets in SOL must be a very rare phenomenon and the incidence of MNs firing doublets calculated in this study as 9.5%, is by no means severely overestimated.

The question which arises from these data is: why doublets were observed in SOL in these two series of experiments? Firstly, these observations were made in experiments of long duration (approximately 1–2.5 h) and never encountered at the beginning of the experiment (see **Figure 5**). This seems to have something in common with "warm-up" phenomenon, i.e., the decrease in the MU firing threshold during repeated or sustained contractions (e.g., Gorassini et al., 2002). This phenomenon has been shown to occur in MNs and was attributed to the facilitation of a voltage-dependent persistent inward current (Svirskis and

Hounsgaard, 1997; Bennett et al., 1998). Recently, similar mechanism was proposed as the explanation for repetitive doublet firing (Kudina and Andreeva, 2010). It may be hypothesized that such a "warm-up" is necessary also for initiation of single doublet generation. In many earlier SOL studies such long sequences might not have been recorded. Moreover, the doublets might often remain unnoticed, if the researcher was analyzing data with another purpose, and/or was not familiar with their specific firing pattern. It is also important to note that the second spike of the doublet usually has different amplitude and/or shape. Recent studies often rely on the automatic recognition software, which may not mark this discharge as belonging to the same MU.

In official EMG terminology (AAEM, 2001), two potentials of the same MU are classified as a doublet, if the interval between them is shorter than 20 ms. However, in this study we report two cases of MUs with intradoublet ISI above 30 ms. The very low variability of these intervals is remarkable, and even more so is their similarity to the triplet ISI, given that they were recorded from two different subjects.

These unusual phenomena are much more seldom than doublets in SOL *per se*, so there is little chance on their detailed investigation in future. We can only propose the explanation for the triplet interval (below), which is highly speculative and should be treated with caution.

It is commonly accepted that doublets recorded in healthy human muscles are generated in MNs exhibiting delayed depolarization (Kudina, 1974; Partanen, 1979; Bawa and Calancie, 1983; Kudina and Churikova, 1990; Kudina and Alexeeva, 1992a; Garland and Griffin, 1999; Piotrkiewicz et al., 2008; Kudina and Andreeva, 2010; Stephenson and Maluf, 2010), which has a shape of a prominent hump that may spontaneously cross the firing threshold, evoking an extra spike (Granit et al., 1963; Kernell et al., 1964; Calvin, 1974). It is possible that certain MNs possess the second hump situated further down the interspike voltage trajectory. This type of hump would be responsible for the third discharge in triplets and for the exceptional doublets, which are generated by MNs without initial hump responsible for usual doublets. The late hump should be very sharp to explain the extremely low variability of triplet and exceptional intradoublet ISIs.

#### **ACKNOWLEDGMENTS**

Sincere thanks are due to Dr. Lydia Kudina for her kind permission to use data recorded in her laboratory and invaluable comments to the manuscript. We also wish to thank the reviewers and the editor for their comments, which significantly contributed to the final version of this paper. All authors were supported by the statutory grants from employing institutions.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 18 September 2013; paper pending published: 10 October 2013; accepted: 19 November 2013; published online: 09 December 2013.*

*This article was submitted to the journal Frontiers in Human Neuroscience.*

*Citation: Piotrkiewicz M, Sebik O, Binboga E, Mło* ˘ *zniak D, Kuraszkiewicz B and* ´ *Türker KS (2013) Double discharges in human soleus muscle. Front. Hum. Neurosci. 7:843. doi: 10.3389/fnhum.2013.00843*

*Copyright © 2013 Piotrkiewicz, Sebik, Binboga, Mło* ˘ *zniak, Kuraszkiewicz and Türker.* ´ *This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Afterhyperpolarization of human motoneurons firing double and triple discharges

#### *Maria Piotrkiewicz\* and Bozenna Kuraszkiewicz ˙*

*Engineering of Nervous and Muscular System, Nałe¸ cz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland \*Correspondence<EMAIL_ADDRESS>
#### *Edited by:*

*Sven Bestmann, University College London, UK*

#### *Reviewed by:*

*Peter Kirkwood, UCL Institute of Neurology, UK*

**Keywords: double discharges, doublets, triplets, afterhyperpolarization, delayed depolarization, motoneurons, human**

#### **INTRODUCTION**

During isometric voluntary contractions of a healthy human muscle, motoneurons (MNs) fire usually with low mean rates, rarely exceeding 25/s (e.g., Garland and Griffin, 1999). However, there are MNs, which sometimes fire double discharges (doublets) with interspike interval (ISI) of few ms. This is observed seldom in normal MNs (e.g., Denslow, 1948; Kudina, 1974; Bawa and Calancie, 1983) but does so more often in neuromuscular disorders (Partanen, 1978; Kostera-Pruszczyk et al., 2002; Piotrkiewicz et al., 2008), where it is considered to be an early sign of MN dysfunction (Rowinska-Marcinska et al., 1999).

Our special attention is paid to the socalled "true" doublets recorded in some MNs under conditions of constant synaptic drive, as opposed to those recorded during rapid contractions (Bawa and Calancie, 1983; Kudina and Andreeva, 2013a). Initial doublets, observed during repetitive movements such as locomotion (Zajac and Young, 1980; Hennig and Lomo, 1987) or respiration (Kirkwood and Munson, 1996), where the synaptic drive undergoes periodic changes, also cannot be classified as "true" doublets. For the sake of clarity, the adjective "true" will be omitted in the further text.

It has been observed that not each human MN is capable of firing doublets (Kudina, 1974; Bawa and Calancie, 1983). By the analogy to animal studies, it was hypothesized that doublets may be generated only in these MNs, which possess the delayed depolarization (DD, Granit et al., 1963; Kernell, 1964; Calvin, 1974) with a prominent hump that may spontaneously cross the firing threshold and evoke an extra spike (Nelson and Burke, 1967; Calvin, 1973). This hypothesis was verified by Kudina and Churikova (1990), who tested the changes in excitability of human motoneurons within ISI by studying the responses of single MNs to the stimulation of Ia afferents. In MNs capable of firing doublets they revealed the period of increased excitability during the first 15 ms after regular discharge, which corresponded well to the duration of DD observed in animal experiments (Granit et al., 1963; Kernell, 1964; Nelson and Burke, 1967; Calvin, 1973).

Nowadays, DD is widely accepted as the mechanism responsible for doublet generation in human MNs (e.g., Kudina, 1974; Bawa and Calancie, 1983; Kudina and Churikova, 1990; Garland and Griffin, 1999). However, it can explain only the origin of occasional doublets, interspersed in regular rhythmic MN activity. Repetitive doublets, i.e., series of doubletpostdoublet ISIs, require some additional mechanism, which would support the DD hump, shown to disappear during MN rhythmic firing (Granit et al., 1963; Calvin and Loeser, 1975). It was recently hypothesized that repetitive doublet firing may be related to plateau potentials (Kudina and Andreeva, 2010, 2013a).

It is still under debate, whether DD is related to other characteristics of a MN, measurable in human experiments. Kernell (1964) noted that DD with a definite hump had a tendency to be more common in MNs with short afterhyperpolarization (AHP), although it could occur with an AHP of any duration. Also in humans, MNs capable of firing doublets were more often found in the fast muscles (Bawa and Calancie, 1983; Kudina and Alexeeva, 1992) than in the slower ones (Andreassen and Rosenfalck, 1980; Kudina and Andreeva, 2013b). For the long time it was believed that the MNs supplying one of the most often investigated slow human muscle, the *soleus*, are devoid of DD. However, it has been recently shown that also MNs supplying human soleus may fire doublets (Piotrkiewicz et al., 2013), which seems to question Kernell's finding. In the present paper we will compare the estimates of AHP duration of MNs capable and not capable of firing double discharges and comment on this controversy. We will also propose a hypothesis on the AHP-related mechanism underlying firing of triplets, which can be observed in some doublet firing MNs.

#### **AHP DURATION OF MNs CAPABLE AND NOT CAPABLE OF FIRING DOUBLE DISCHARGES**

Our opinion is essentially based on the results of the experiments reported earlier (Piotrkiewicz et al., 2001), which were aimed toward investigation of AHP duration in slow (*soleus,* SOL) and fast *(biceps brachii,* BB) human muscle (Exp. 1). Within the motor unit potential trains, recorded in these experiments during steady-state voluntary isometric muscle contractions, we found also doublets and triplets (**Figure 1A**, Piotrkiewicz et al., 2008). Additionally, we included the results from the *triceps brachii* (TB) from another study, designed specifically for the investigation of double discharges (Kudina and Andreeva, 2010). In this study, the subjects were trained to search for doublet firing motor units. They were instructed to slowly develop a gentle voluntary isometric muscle contraction until a motor unit

started firing doublets, and then to keep contraction constant for a few minutes (Exp. 2).

It is commonly accepted that each motor unit discharge is equivalent to the discharge of its MN. Motoneuron AHP duration was estimated from single motor unit potential trains by the method based on the observation of Person and Kudina (1972) that the relationship between the standard deviation and mean value of ISI sharply changes after a certain transition value that was hypothesized to correlate with the AHP duration of the MN. The hypothesis was verified by computer simulations (Piotrkiewicz, 1999) and in the direct recordings from cat MNs (Powers and Binder, 2000), which confirmed this correlation. The AHP duration estimates, which this opinion is based upon, were obtained by the modification of the above method (Piotrkiewicz et al., 2012), using the ISI variability analysis proposed by Holt et al. (1996). The variability was measured as the absolute consecutive difference (CD) between two adjacent ISIs. It was plotted against mean ISI, calculated from the same two intervals (MISI). An example of the plot *CD* vs. MISI is shown in **Figure 1B** (orange dots). The mean *CD* values (*CDm*, green squares) were calculated after grouping the MISIs in 10 ms bins with 5 ms overlap. After rejection of points calculated from insufficient number of *CD* values, the mean data were fit with two linear regression lines and the intersection of these lines used to determine transition ISI, being an estimate of AHP duration.

The AHP duration was estimated for 80 MNs from BB, 90 MNs from SOL, and 28 MNs from TB. From these, MNs firing doublets constituted 5% for BB (4 MNs), 9% for SOL (8 MNs), and 64% for TB (18 MNs). Two MNs recorded in Experiment 1, one from BB and one from SOL, fired also triplets that were much scarcer than doublets: for BB MN 11 triplets vs. 1592 doublets, for SOL MN 6 triplets vs. 342 doublets. **Figure 1A** (right) presents characteristic triplet firing pattern, which was similar in the two MNs: the ISI between first two components was equal to the doublet ISI, whereas triplet ISI measured between second and third spike was substantially longer (2.4 times for BB and 6.5 times for SOL).

**Figure 1C** shows the comparison of transition ISIs for MNs with (filled symbols) and without doublets (open symbols) from BB, SOL, and TB. The ranges of both clusters overlap, but the mean values of transition ISIs calculated in each of MN pools of BB and SOL separately are shorter for doublet firing MNs than for MNs without doublets: 85.70 vs. 99.39 and 128.01 vs. 148.04 ms, respectively. Thus, although the AHPs in doublet firing MNs supplying SOL are longer than in their counterparts supplying BB, within the pool of each muscle they tend to be shorter than those in the other MNs.

At the first sight, TB data do not seem to confirm this observation. The difference in mean values of transition ISIs between MNs with and without doublets is negligible: 80.40 vs. 80.00 ms and many of MNs from the former group present transition ISIs longer that those from the latter. However, there are striking differences between TB and BB in doublet incidence and transition ISI ranges. Since both TB and BB are fast muscles, it could be presumed that the AHP duration ranges of their MNs should be comparable. Moreover, it should be noted that the contraction strengths in the Exp. 2 were lower than in the Exp. 1 (below 10 and 30% maximum voluntary contraction, respectively). Thus, the MNs recorded from TB could be expected to have the lower thresholds than those from BB. Consequently, according to the commonly known rules of the orderly recruitment (Milner-Brown et al., 1973a,b; Henneman et al., 1974) as well as to the match observed between motor unit twitch contraction time and the AHP duration of its MN (Kernell et al., 1999), one could expect that AHPs estimated for TB MNs would be rather longer than those of BB MNs. Despite these expectations, the transition ISIs in TB not only are shorter, but their range is much narrower than that of BB MNs.

The differences observed could be related to the differences in experimental protocols, specifically to the different MN sampling. Whereas the sampling in the Exp. 1 could be assumed to be random, in Exp. 2 it was influenced by the training for search of MNs with doublets. It is impossible to say, what happens with MNs during training. It must involve some changes in synaptic inflow, since the majority of doublets recorded in Exp. 2 were repetitive ones. However, it may also be presumed that due to this training the MN sample was limited to those cells, in which finding hump-like DD was most feasible, i.e., to the MNs with shortest AHPs. The narrow range of transition ISIs and the very high proportion of doublet firing MNs in TB sample (64%) are in favor of this presumption. Summing up, the results from all three muscles seem to be in line with the earlier observation of Kernell (1964).

#### **INTERSPIKE INTERVALS OF DOUBLE AND TRIPLE DISCHARGES vs. AHP DURATION**

**Figure 1D** presents the dependency of the intervals between spike components of doublets and triplets on the AHP duration. The doublet ISI duration of MNs supplying all muscles investigated was independent of transition ISI (open symbols). This result is in line with the suggestions of Granit et al. (1963) and Kernell (1964) that DD may not be the "true" afterpotential. The more recent studies on the ion channels involved in the generation of MN rhythmic firing confirm that the mechanisms responsible for AHP and DD differ from each other (Viana et al., 1993; Kobayashi et al., 1997).

In contrast to the doublet ISI, the triplet ISI measured for SOL (blue filled square) was substantially longer than that measured for BB (red filled square). This observation may suggest that its firing might not be caused by DD, but by the later hump in the time course of AHP conductance described by Baldissera and colleagues (Baldissera and Gustafsson, 1974; Baldissera and Parmiggiani, 1979). This hump has been shown to coincide with the AHP time-to-peak, which would be longer in MNs with longer AHPs. Moreover, AHP summation after short doublet ISI may enhance conductance hump. On the other hand, AHP summation would result in diminished DD (Granit et al., 1963), which also indicates that the firing of the third spike related to DD is hardly possible.

#### **CONCLUDING REMARKS**

Doublets are rarely recorded during the voluntary muscle activity in healthy human subjects. They are generated by the MNs with hump-like DD, whose characteristics, which could be measured in human experiments, are largely unknown. However, the knowledge of the properties of these MNs is important, since their incidence increases significantly in some neuromuscular diseases, where it is considered to be an early sign of MN dysfunction. We believe that although AHPs of doublet firing MNs supplying SOL are longer than those of BB and TB, in each MN pool the doublet firing human MNs belong to those with shortest AHPs, as do the cat MNs with hump-like DDs. We admit, however, that by now the evidence supporting this belief is rather weak.

Triplets are much more scarce than doublets. Thus, collecting bigger experimental material is virtually impossible. We decided to present this opinion hoping that it may inspire other researchers to look for these rare events in their experimental recordings. We are aware that our data are not sufficient to justify solid conclusions concerning mechanism(s) underlying triplet firing. Nevertheless the specificity of the opinion article encouraged us to formulate the above presented speculative hypothesis about the unusual, intriguing phenomenon of triplet firing.

#### **ACKNOWLEDGMENTS**

The sincere thanks are due to Drs. Lydia Kudina and Regina Andreeva for kind permission to use their experimental data as well as for valuable comments during manuscript preparation. We are also indebted to the Referee, whose deep criticism helped to make this paper more comprehensive. The authors were supported by the statutory grant from IBBE PAS.

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 February 2014; accepted: 14 May 2014; published online: 30 May 2014.*

*Citation: Piotrkiewicz M and Kuraszkiewicz B (2014) Afterhyperpolarization of human motoneurons firing double and triple discharges. Front. Hum. Neurosci. 8:373. doi: 10.3389/fnhum.2014.00373*

*This article was submitted to the journal Frontiers in Human Neuroscience.*

*Copyright © 2014 Piotrkiewicz and Kuraszkiewicz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Testing the excitability of human motoneurons

#### *Chris J. McNeil 1,2, Jane E. Butler 1,3, Janet L. Taylor 1,3 and Simon C. Gandevia1,3\**

*<sup>1</sup> Neuroscience Research Australia, Randwick, NSW, Australia*

*<sup>2</sup> School of Health and Exercise Sciences, University of British Columbia, Kelowna, BC, Canada*

*<sup>3</sup> Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia*

#### *Edited by:*

*Ulf Ziemann, Eberhard Karls University Tübingen, Germany*

#### *Reviewed by:*

*Ulf Ziemann, Eberhard Karls University Tübingen, Germany Yoshikazu Ugawa, Fukushima Medical Universtiy, Japan*

#### *\*Correspondence:*

*Simon C. Gandevia, Neuroscience Research Australia, PO Box 1165, Randwick, NSW 2031, Australia. e-mail<EMAIL_ADDRESS>
The responsiveness of the human central nervous system can change profoundly with exercise, injury, disuse, or disease. Changes occur at both cortical and spinal levels but in most cases excitability of the motoneuron pool must be assessed to localize accurately the site of adaptation. Hence, it is critical to understand, and employ correctly, the methods to test motoneuron excitability in humans. Several techniques exist and each has its advantages and disadvantages. This review examines the most common techniques that use evoked compound muscle action potentials to test the excitability of the motoneuron pool and describes the merits and limitations of each. The techniques discussed are the H-reflex, F-wave, tendon jerk, V-wave, cervicomedullary motor evoked potential (CMEP), and motor evoked potential (MEP). A number of limitations with these techniques are presented.

**Keywords: motoneuron, H-reflex, F-wave, tendon jerk, V-wave, CMEP, MEP**

#### **INTRODUCTION**

The motoneuron was described as the "final common path" of the nervous system (Sherrington, 1906) and has been a focal point of neuroscience research for over a century. Since the introduction of the terminology, the designation of "final common path" has been frequently expanded to include not only the α-motoneuron but also the muscle fibers which it innervates (i.e., the motor unit) (e.g., Denslow and Hassett, 1942). This expanded definition is sensible from a functional perspective because movement requires contraction of muscle fibers and the properties of muscle fibers are largely dictated by the properties of the motoneuron which innervates them (see Burke, 1981; Henneman and Mendell, 1981; Binder et al., 1996 for reviews).

Motor unit properties have been directly studied in animals (e.g., Burke et al., 1971; Peter et al., 1972) and are directly related to the size of the motoneuron. In brief, motoneurons with a large soma (and hence a large axonal diameter) have a low input resistance, high firing threshold, brief after-hyperpolarization, fast conduction velocity, and their muscle fibers have a fast twitch contraction time, high twitch tension and a poor resistance to fatigue. As the size of motoneurons decreases, these responses gradually shift to the opposite ends of their spectra. Output properties of human motoneurons can be gleaned from wholenerve stimulation, spike-triggered averaging (e.g., Milner-Brown et al., 1973; Thomas et al., 1990) or intraneural stimulation (e.g., Thomas et al., 1990). Measurement of somato-dendritic properties is more difficult and frequently relies on reflex and antidromic inputs to the motoneurons.

Like the assessment of motor unit properties, tests of motoneuron pool excitability in humans are necessarily indirect. Before we discuss the most common methods used to test excitability of the human motoneuron pool, it is first necessary to define our use of the term "excitability" in this review. For our purposes, the term "excitability" is a relative one. That is, if the same input is delivered to the motoneuron pool before and after an intervention (e.g., muscle fatigue), do more or fewer motoneurons generate action potentials after the intervention? A bigger or smaller output would represent a net increase or decrease in motoneuron excitability, respectively. The change in excitability will reflect the balance of inhibition and facilitation but it is difficult to determine the mechanism in a given situation; e.g., a decrease in excitability could be due to either an increase in inhibition or a decrease in facilitation. Further, any change in "excitability" may not apply uniformly across the whole motoneuron pool.

Regardless of the methodology or the intervention studied (e.g., acute vs. chronic), the goal of testing human motoneuron excitability is the same: to know more about the status of the motoneuron pool. The aim of this review is to discuss briefly the existing methodologies which test motoneuron excitability by evoking compound muscle action potentials with particular focus on the benefits and limitations of each. Discussion of the H-reflex, F-wave, tendon jerk, V-wave, cervicomedullary motor evoked potential (CMEP) and motor evoked potential (MEP) is included below and summarized in **Table 1**. There are a number of single motor unit approaches with their own advantages and disadvantages but these methods are largely beyond the scope of this review. However, it deserves emphasis that these approaches provide useful information but usually only about a limited number of motoneurons which have a low-threshold in a voluntary contraction.

#### **H-REFLEX**

Stimulation of a peripheral nerve can evoke a reflex response termed the Hoffmann or H-reflex (Magladery and McDougal, 1950) because it was first described in the soleus muscle by Hoffmann (1918). The H-reflex reflects the response of the motoneuron pool to a volley from large-diameter primary muscle

*Note: Submaximal/supramaximal refers to the stimulus strength relative to the current required to evoke the maximal compound muscle action potential (Mmax ).*

spindle afferents (**Figure 1A**). Most commonly recorded as a multi-unit response from surface electromyographic activity, it is possible to record an H-reflex in single motor units (e.g., Trontelj, 1968; Ashby and Zilm, 1982; Burke et al., 1984; Miles et al., 1989). Similar to the descending input during voluntary contractions, the synaptic Ia input will recruit motoneurons in an orderly fashion from smallest to largest (slow to fast motor units; Buchthal and Schmalbruch, 1970) according to the Henneman

for a complete description of the factors which can influence each response. **(A)**, #1—electrical stimulus evokes a single afferent volley which recruits motoneurons for the H-reflex according to the size principle; #2—presynaptic inhibition can influence afferent input to the motoneuron; #3—tendon tap evokes multiple volleys which arrive at the motoneuron over 25 ms. **(B)**, #1—a small number of motoneurons collision with the antidromic volley prior to the soma. **(C)**, #1—in motor axons conducting orthodromic impulses of voluntary drive, voluntary and antidromic impulses will collide; #2—reflex response which travels along motor axons cleared by the collision described in point #1 will contribute to the V-wave; #3—presynaptic inhibition can influence afferent input to the motoneuron.

size principle (e.g., Henneman and Mendell, 1981). Delivery of a series of progressively stronger stimuli generates a recruitment curve of the H-reflex and the muscle compound action potential (M-wave). In brief, H-reflex size increases with stimulus intensity until it reaches a maximum. This point can occur when further increases in intensity do not result in further increases in the net excitatory input to the motoneurons. Alternatively, a maximum can be reached because further increases in intensity reduce H-reflex size due to collision of afferent-evoked orthodromic impulses with antidromic impulses evoked in the motor axons that contribute to the growing M-wave. The H-reflex recorded at the tipping point is referred to as *H*max. Comparison of Hreflex size to the maximal M-wave enables an estimate of the motoneuron pool involved in the H-reflex. For the soleus muscle in most subjects the percentage of involved motoneurons is ∼50% (Taborikova and Sax, 1968). A recruitment curve provides additional parameters (e.g., H-reflex threshold, slope of the ascending limb of the recruitment curve) which give insight into H-reflex input/output relationship (e.g., Zehr, 2002; Klimstra and Zehr, 2008). Finally, data about recruitment curves improve the validity of comparisons of data across time or experimental conditions.

Initially believed to be a purely monosynaptic Ia reflex (Hoffmann, 1922; Magladery et al., 1951; Paillard, 1955), it has since been established that the relatively long rise time of the compound excitatory postsynaptic potential (EPSP) (1.9–2.1 ms in soleus motoneurons; Birnbaum and Ashby, 1982; Burke et al., 1983) enables disynaptic (and possibly oligosynaptic) Ia pathways and Ib afferents to exert an influence on the H-reflex (Burke et al., 1983, 1984). Because the threshold to electrical stimulation and the conduction velocity of the fastest Ia and Ib afferents are probably not greatly different, both afferents feature prominently in the initial volley which arrives at the spinal cord (Pierrot-Deseilligny et al., 1981). Ib afferents acting via an inhibitory interneuron do not prevent the monosynaptic Ia EPSPs which initiate the H-reflex but do terminate the EPSPs with inhibitory postsynaptic potentials (IPSPs) at an interval as brief as 1 ms (Pierrot-Deseilligny et al., 1981). The first experimental, rather than theoretical, evidence of this non-reciprocal group I inhibition was a disynaptic limitation of the size of the quadriceps H-reflex (Marchand-Pauvert et al., 2002). Although this inhibition limits the size of the H-reflex (Burke et al., 1984), the majority of the response recorded under most conditions reflects monosynaptic Ia afferent input to the motoneuron pool. The influence of disynaptic or oligosynaptic input on motoneuron recruitment is determined by the size of the compound monosynaptic EPSP relative to the recruitment threshold of each motoneuron. That is, early recruited units in the response will be recruited by monosynaptic input but the discharge of the last recruited motoneurons will reflect the balance between monosynaptic excitation and dior oligosynaptic inhibition and/or excitation. Hence, a change in H-reflex size occurs primarily through a change in this balance. This is true regardless of the size of the test H-reflex.

For more than 50 years, the H-reflex has been widely used as a test of the excitability of the human motoneuron pool. However, there are a number of caveats for this test which are commonly ignored or under-appreciated, despite a wealth of experimental data (e.g., Paillard, 1955) and detailed discussion of the technique (e.g., Schieppati, 1987; Pierrot-Deseilligny and Mazevet, 2000; Zehr, 2002; Pierrot-Deseilligny and Burke, 2005). A major mechanism that has long been known to alter the size of the H-reflex is the degree of presynaptic inhibition of Ia terminals (e.g., Frank and Fuortes, 1957; Eccles et al., 1961; Hultborn et al., 1987; see Rudomin and Schmidt, 1999 for review). Some other key mechanisms which can influence the size of the H-reflex include post-activation depression or homosynaptic depression (e.g., Magladery and McDougal, 1950; Crone and Nielsen, 1989; see Hultborn and Nielsen, 1998 for review) and contributions of oligosynaptic pathways (e.g., Pierrot-Deseilligny et al., 1981; Burke et al., 1983, 1984). Finally, in testing the H-reflex, it is difficult to be sure that the afferent volley itself is constant because activity leads to axonal hyperpolarization and reduced excitability in sensory and motor axons (e.g., Kiernan et al., 1997; Vagg et al., 1998) such that the same stimulus intensity is likely to activate fewer afferent axons after a voluntary contraction. See Pierrot-Deseilligny and Burke (2005) for a detailed discussion of these and other mechanisms.

The size of an H-reflex is sensitive to changes in subject posture (Hugon, 1973) and attention (Bathien and Morin, 1972), so it is critical that these factors vary as little as possible when collecting H-reflexes. Further, these factors make day-to-day comparisons of the H-reflex particularly difficult. The H-reflex is also strongly influenced by the frequency of stimulation as postactivation depression reduces the size of a second response elicited within 10 s of the first. To avoid this reflex attenuation, the stimulation frequency of repeated H-reflexes would ideally not exceed 0.1 Hz. However, collection of the requisite large number of responses at this rate is time-consuming and so a faster stimulation rate is more practical even if some post-activation depression remains. As the decay of post-activation depression is curvilinear (Magladery and McDougal, 1950), stimulating at 0.2–0.3 Hz strikes an acceptable balance between the level of depression and the time required to collect the responses (Pierrot-Deseilligny and Mazevet, 2000). The frequency of stimulation can be increased as high as 4 Hz during voluntary contraction because the postactivation depression seen in relaxed muscle is greatly attenuated or abolished (Burke et al., 1989; see also Stein et al., 2007) possibly because the extra impulse evoked by the electrical stimulus will have negligible impact on transmitter release from Ia afferents which are already discharging (Stein and Thompson, 2006). Although the size of the effect was relatively small, another factor to consider is the regularity of the stimuli. An interstimulus interval which varied between 0.5 and 1.5 s (mean of 1 s) evoked a larger H-reflex compared to stimulation at a constant interval of 1s(Hoehler et al., 1981).

A practical limitation of H-reflexes is that they can only be obtained consistently from a small number of muscles during relaxation (typically soleus, flexor carpi radialis, quadriceps). However, this list of muscles expands greatly if stimulation is given while the subject performs a weak voluntary contraction. This ensures some motoneurons are discharging repetitively, brings others closer to threshold and thus increases motoneuron excitability. Burke and colleagues (1989) identified a number of other benefits to testing the H-reflex during voluntary contraction which include: the abolition of post-activation depression (homosynaptic depression); larger response sizes with lower stimulus intensities and hence a clearer distinction between the end of the M-wave and the onset of the H-reflex; a focus of the reflex response to the active motoneuron pool so that specific reflex arcs can be studied. Additional benefits are a reduction in the levels of homonymous (Fournier et al., 1983) and heteronymous Ib inhibition (Pierrot-Deseilligny and Fournier, 1986). Presynaptic inhibition is reported to be the same at rest and during steady contraction (Meunier and Pierrot-Deseilligny, 1989; Nielsen and Kagamihara, 1993) and so appears to represent neither an advantage nor a disadvantage to testing the H-reflex during voluntary contraction.

Thus, far, this section has described homonymous connections, that is from the stimulated Ia afferents to the motoneuron pool of the same muscle. However, there are also heteronymous links between Ia afferents of one nerve to the motoneuron pools of muscles supplied by a different nerve. For example, stimulation of the femoral nerve delivers monosynaptic Ia excitation not only to the quadriceps motoneurons but also to the soleus motoneuron pool (e.g., Bergmans et al., 1978; Meunier et al., 1990). Hence, appropriately-timed stimulation of the femoral nerve facilitates the soleus H-reflex. Heteronymous Ia monosynaptic excitation has been demonstrated in both the upper (e.g., Cavallari and Katz, 1989; Marchand-Pauvert et al., 2000) and lower limbs (e.g., Meunier et al., 1993) of humans. The utility of these connections is the ability to test the excitability of a motoneuron pool without the serious effect of activity-dependent changes in the excitability of the homonymous afferents.

### **F-WAVE**

Although not termed the F-wave until 1950 (Magladery and McDougal, 1950), this late response to stimulation of a peripheral nerve was first described by Eccles and Pritchard (1937). Described as a recurrent discharge (e.g., Eccles, 1955), the F-wave reflects backfiring of a small number of motoneurons which are reactivated by antidromic impulses following supramaximal stimulation of a peripheral nerve (**Figure 1B**). Because F-waves are small (often less than 0.5 mV) and inconsistent in both size and shape, large numbers of responses are collected for averaging (Lin and Floeter, 2004). The variability of onset latency and morphology would cause considerable phase cancellation if raw F-waves were averaged online so potentials must be measured individually or full-wave rectified prior to averaging (Espiritu et al., 2003). In a clinical setting, rather than calculating the average response to a large number of stimuli, persistence (the percentage of stimuli evoking a response) and the difference in latency between the onset of the slowest and fastest single motor unit potentials are used as objective measures of properties of the motoneuron pool.

The production of an F-wave by a given motoneuron is believed to depend on the excitability of the axon initial segment (Eccles, 1955), and perhaps also the first node of Ranvier (Gogan et al., 1984). The passage of the antidromic impulse to the soma will make these sites transiently refractory. If the axon initial segment remains refractory when the antidromic impulse evokes a somato-dendritic action potential, an orthodromic action potential will not be initiated in the axon to be propagated to the periphery and recorded as an F-wave. Intraneural stimulation of single thenar motor axons indicates that generation of an F-wave in an individual motoneuron is probabilistic and occurs rarely (after *<*2% of stimuli). Further, their incidence is unrelated to motor axon conduction velocity or twitch force (Thomas et al., 2002). This suggests a contribution from a mixed population of motoneurons. However, in more common experimental and clinical testing, the stimulus intensity is higher and the motoneurons which generate F-waves are likely to be limited to large motoneurons due to reflex activation of smaller motoneurons and collision with the antidromic volley prior to the soma (Espiritu et al., 2003).

Interpretation of the F-wave is simplest when the muscle is relaxed at the time of stimulation but responses can be recorded during voluntary contraction (e.g., Giesebrecht et al., 2011). The disadvantage to collecting responses during contraction is that a collision between voluntary orthodromic impulses with antidromic impulses will leave some motor axons clear to transmit an H-reflex to the muscle and thereby obscure the F-wave. This problem is exacerbated as the strength of contraction increases because a greater proportion of motor axons will see their antidromic impulse obliterated before it reaches the soma. During strong voluntary contractions, few antidromic impulses will reach the soma because of collision with the orthodromic voluntary and reflex action potentials. In these circumstances, any recorded potential would almost certainly be a V-wave (see V-wave section which follows).

It has been suggested that F-waves are a useful and direct measure of motoneuron excitability (e.g., Fisher, 1992) but subsequent reports suggest that F-waves only offer a flawed measurement of motoneuron excitability (Hultborn and Nielsen, 1995; Espiritu et al., 2003; Lin and Floeter, 2004; Pierrot-Deseilligny and Burke, 2005). Chief among the limitations is the relative insensitivity of F-waves to changes in motoneuron excitability. Nonetheless, they are potently depressed following fatiguing voluntary contractions (Khan et al., 2012; Rossi et al., 2012). A reduction in F-waves can be caused not only by inhibition of the motoneuron pool but by facilitation as well (Eccles, 1955; Hultborn and Nielsen, 1995). In a strongly facilitated motoneuron pool, the antidromic impulse which invades the soma will be followed by a somato-dendritic action potential at such a short interval that the axon initial segment is still refractory (Eccles, 1955). However, this may not be a practical limitation in human studies based on results with voluntary contractions (Giesebrecht et al., 2011). The other key criticisms of the F-wave concern the practice of comparing H-reflexes and F-waves in an effort to separate events at the level of the motoneurons; that is, to gain insight into changes in presynaptic inhibition versus changes in motoneuron excitability (e.g., Leis et al., 1995). Hultborn and Nielsen (1995) questioned the validity of such a comparison on three theoretical bases. The first relates to the collision between antidromic impulses and H-reflex discharges in slowly conducting motor axons. An enhancement of motoneuron excitability (e.g., by voluntary contraction) will increase the size of the H-reflex and thereby actually decrease the number of motoneurons capable of producing an F-wave because of a greater number of collisions. Second, the motor unit populations involved in the two potentials differ. The H-reflex involves small motor units with slowly conducting axons whereas, for reasons described above, the F-wave is likely to involve large motor units with fast axons. The third point relates to the mode of activation of the two responses (afferent vs. antidromic). To illustrate their concerns, Hultborn and Nielsen (1995) conducted a simple experiment which showed that F-waves could be an order of magnitude less sensitive than H-reflexes to changes in motoneuron excitability (although both responses were facilitated by a conditioning stimulus to the femoral nerve).

#### **TENDON JERK**

For limb muscles in which an H-reflex is not easily obtained, an alternative method of testing motoneuron excitability is a tendon jerk reflex. A tendon tap with a reflex hammer or more controlled means will create a stretch-induced barrage of discharges from muscle spindle primary endings and other afferents (**Figure 1A**). The size of the muscle spindle afferent volley depends on the mechanics of the tap and the sensitivity of the sensory endings. This sensitivity can be changed by contraction of the intrafusal muscle fibers on which the endings are located. Like the electrically-induced H-reflex, the mechanically-induced tendon jerk cannot be considered purely monosynaptic (e.g., Burke et al., 1983). However, unlike the relatively synchronous volley of the H-reflex, the afferent volley of the tendon jerk includes multiple discharges of a single Ia afferent and lasts for 25 ms (Burke et al., 1983, 1984). As a result, the afferent volley which arrives at the motoneurons is more dispersed for the tendon jerk than the H-reflex. This is one of several differences between the two reflexes which invalidate a comparison of the two responses as a surrogate measure of efferent drive to the muscle spindles (i.e., fusimotor activity) (Burke et al., 1983).

While ongoing fusimotor drive must potentially alter the size of the afferent volley evoked by a tendon tap, the history of prior fusimotor drive exerts a potent effect on the volley (Polus et al., 1991)due to the "thixotropic" behavior of the intrafusal muscle fibers (see Proske et al., 1993 for review). If a muscle is undisturbed or lengthened slowly following fusimotor activation, intrafusal fibers remain taut as actin-myosin cross bridges are maintained or re-established in a more stretched position. Conversely, passive shortening after fusimotor activation will maintain the cross bridges but cause the intrafusal fibers to become slack (Polus et al., 1991). This can produce large changes in background spindle firing rates. When spindles are held taut as a result of prior activity, the response to a tendon tap is increased. Note that this will have the opposite effect on the H-reflex as the increased background firing will lead to increased presynaptic inhibition (Polus et al., 1991).

The long held view that background fusimotor drive is necessary to elicit a tendon jerk from relaxed muscle is false (Burke et al., 1981). The relative ease of obtaining a tendon jerk in one muscle compared to another is likely to depend on intrinsic spinal mechanisms. While the tendon jerk has the advantage of being simple to administer, changes in its size depend on many factors, beginning with the mechanical status of the tendon and transmission of the transient lengthening to the receptors.

#### **V-WAVE**

First described by Upton and colleagues (1971), the volitional wave (V-wave) is a variation of the H-reflex which is recorded during a voluntary contraction. In contrast to the submaximal stimulation used to evoke an H-reflex (see H-reflex section), a V-wave is evoked by supramaximal stimulation of a peripheral mixed nerve (**Figure 1C**). The supramaximal stimulus generates antidromic impulses in all motor axons as well as impulses in all group Ia afferents provided the stimulus intensity is ≥ 4× motor threshold (Gracies et al., 1994). In motor axons involved in the voluntary contraction, there is a collision between voluntary orthodromic and the evoked antidromic impulses which leaves these axons clear to transmit a reflex response to the muscle. Conversely, motor axons not involved in the contraction, do not contribute to the production of the V-wave because any reflex response will either collide with the antidromic impulse or the antidromic impulse will reach the soma first and leave these motoneurons refractory when the afferent volley arrives.

The V-wave is influenced by many factors (e.g., strength of voluntary contraction and the range of maximal firing rates within a motoneuron pool) and the consequent difficulty in interpreting a change to the response raises questions about its usefulness as an independent measure of motoneuron excitability. During a maximal voluntary contraction, the size of the V-wave is proposed to indicate the level of descending voluntary drive conveyed by the motoneurons (Aagaard et al., 2002). According to this proposal, an increase in V-wave size indicates increased motoneuron discharge rates or recruitment (Aagaard et al., 2002) which may reflect increased supraspinal input to the motoneuron pool. However, it is important to recall that motoneuron discharge rate reflects not only supraspinal input to the motoneuron but the response to all inputs which arrive at the motoneuron so the origin of an increase in V-wave size is uncertain. The possible sensitivity of the V-wave to supraspinal input is unlike that of the H-reflex which is largely dependent on events at a spinal level (see H-reflex section). This distinction has led to the recent application whereby changes in the V-wave are compared to those in the H-reflex before and after training to distinguish between supraspinal and spinal neural adaptations (Aagaard et al., 2002; Vila-Cha et al., 2012).

#### **CMEP**

Non-invasive electrical or magnetic stimulation of spinal tracts can evoke large, short-latency responses in arm and leg muscles (Ugawa et al., 1991, 1994; Gandevia et al., 1999; Martin et al., 2008; see Taylor and Gandevia, 2004 for review). To evoke responses in arm muscles, axons are activated at the level of the cervicomedullary junction near the pyramidal decussation (**Figure 2**). Consequently, such a response is generally termed a CMEP. Electrical stimulation is accomplished by passing a

brief high-voltage pulse between electrodes fixed near the mastoid processes. For magnetic stimulation, a double-cone coil is placed at the back of the head, with the center of the coil near the inion. Several terms describe these forms of stimulation and they are often used interchangeably. These include cervicomedullary, transmastoid, brainstem, or corticospinal tract stimulation. However, because the stimulus site can vary, not all the terms are actually synonymous. For example, responses with corticospinal components can be obtained in leg muscles with electrical stimulation over the cervical or thoracic spine (Martin et al., 2008). Responses produced by stimulation over the thoracic spine, are referred to as thoracic spine MEPs (TMEPs; Martin et al., 2008) rather than CMEPs.

Descending motor pathways other than the corticospinal tract (as well as ascending pathways) will be activated by the stimulus, but there is strong evidence that the CMEP is primarily the result of motoneuron activation by a single descending volley elicited by excitation of corticospinal axons (Berardelli et al., 1991; Ugawa et al., 1991; Gandevia et al., 1999; Taylor et al., 2002). The singular nature of the descending volley was confirmed by epidural recording in anaesthetized patients (Rothwell et al., 1994). In awake subjects, a collision experiment in which an ulnar nerve stimulus given before the brainstem stimulus (Berardelli et al., 1991) caused complete occlusion of the CMEP in abductor digiti minimi. In contrast, an ulnar nerve stimulus did not fully occlude the response to transcranial electric (Day et al., 1987) or magnetic stimulation (Hess et al., 1987) of the motor cortex which induce multiple corticofugal volleys and multiple discharges from some motoneurons. Additional collision experiments demonstrate that the CMEP is largely conducted via the large-diameter axons of the corticospinal tract. Brainstem stimulation given at appropriate times relative to electrical (Ugawa et al., 1991) or magnetic stimulation of the motor cortex (Berardelli et al., 1991; Gandevia et al., 1999; Taylor et al., 2002) largely occluded the motor cortical evoked potential (MEP) which suggests that the two stimuli activate many of the same corticospinal axons.

There are two important attributes of CMEPs which make this stimulation technique the most direct method to test motoneuron excitability to synaptic input in conscious humans (Martin et al., 2008). First, there is evidence that they have a large monosynaptic component in the upper limb (Petersen et al., 2002) and probably in the lower limb (Martin et al., 2008). Second, the descending tracts are not subject to conventional presynaptic inhibition due to primary afferent depolarization (Nielsen and Petersen, 1994; Jackson et al., 2006). This latter feature is in contrast to the H-reflex pathway (see Rudomin and Schmidt, 1999 for review). However, CMEPs must also be influenced by non-monosynaptic inputs although these have not been identified, and changes in CMEPs after strong voluntary contractions are postulated to reflect some presynaptic mechanism other than conventional presynaptic inhibition (Gandevia et al., 1999). Another advantage of the CMEP is that large responses can be evoked and hence averaging of large numbers of responses is not usually necessary.

Despite the advantages of corticospinal stimulation, like all stimulation techniques, it has limitations. A practical disadvantage is the discomfort produced by the stimulus. Most subjects will tolerate the stimuli but some find them prohibitively painful. The issue of pain is particularly relevant for stimuli delivered while the subject is relaxed because the discomfort is much less during muscle contraction and decreases as the level of voluntary effort increases. Apart from the issue of greater transient discomfort for the subject, the pain of stimulation when the subject is relaxed can indirectly affect the data. The size of the CMEP is sensitive to motoneuron excitability and so data collected in relaxation can be contaminated by weak inadvertent contraction if the subject instinctively "tenses up" in anticipation of the stimulus.

Another disadvantage of corticospinal stimulation is the inability or difficulty in obtaining responses of sufficient size in some subjects and some motoneuron pools. Even in subjects who tolerate stimuli within the normal range of intensities, it may not be possible to activate motoneurons via descending tract stimulation and record a valid CMEP. This occurs when the stimulus intensity required to evoke a response also activates nerve roots distal to the motoneuron soma. Such direct activation of the motor axon will mean that the "CMEP" is contaminated by a direct motor response and may not reflect motoneuron excitability accurately. The presence of nerve root stimulation can be identified in two ways: an abrupt ∼1–2 ms reduction in onset latency of the CMEP with an increase in stimulus intensity; or the absence of a large increase in CMEP size (relative to the CMEP recorded in relaxation) if a given stimulus is delivered during a weak voluntary contraction.

Corticospinal (transmastoid) stimulation has recently been paired with transcranial magnetic stimulation (TMS) as a novel means to test motoneuron responsiveness during ongoing muscle activity and fatigue without the confounding influence of unknown levels of descending voluntary drive (McNeil et al., 2009, 2011a,b,c). With this technique, a corticospinal stimulus is delivered 100 ms after a strong conditioning TMS pulse which transiently (∼200 ms) silences descending drive. The interruption of descending drive briefly stops motoneuron output so the excitability of motoneurons can be tested in a state of artificial relaxation (McNeil et al., 2009) without stopping the task and thereby altering the progression of fatigue.

#### **MEP**

TMS of the motor cortex (**Figure 2**) is widely used to test "motor cortical" excitability but is included here because of the profound impact of motoneuron excitability on the size of the MEP. This effect is best demonstrated by the comparison of MEPs recorded from a muscle during relaxation and voluntary contraction. Regardless of stimulus intensity (e.g., Di Lazzaro et al., 1998; McNeil et al., 2011a), MEP size increases markedly from relaxation to a weak contraction and the principal mechanism for this shift is enhanced motoneuron excitability (Hess et al., 1987; Taylor et al., 1997; Di Lazzaro et al., 1998). Hence, researchers must exercise caution when interpreting changes in MEP size as changes in "cortical" excitability. To make this claim, a valid test of motoneuron excitability must be performed to eliminate the possibility that the change in MEP size is mediated at a motoneuronal level. Even then, changes in MEP size may not represent changes in "cortical" excitability as there are significant non-monosynaptic contributions to MEPs so that changes at premotoneuronal sites can modify MEP size. For example, for muscles of the upper limb other than intrinsic hand muscles, significant excitation occurs through the C3–4 propriospinal system (Gracies et al., 1991).

#### **CONCLUSIONS**

One element of testing motoneuron excitability which needs further investigation is the matter of specificity. That is, if an increase or decrease in excitability is noted with one method (e.g., CMEP), is the same change evident when other methods (e.g., H-reflex) are applied? We recently compared the effects of fatigue on CMEPs (TMEPs) and F-waves (Giesebrecht et al., 2011) and there are the previously described comparisons between H-reflexes and F-waves (Hultborn and Nielsen, 1995) and H-reflexes and V-waves (Aagaard et al., 2002; Vila-Cha et al., 2012). However, additional comparisons of this nature would increase our understanding of the mechanisms involved and provide insight into the validity of different methods under various conditions.

 *Ann. Neurol.* 10, 547–550.

production of voluntary isolated movements in man. *Brain Res.* 288, 375–377.

Assessing changes in presynaptic inhibition of I a fibres: a study in man and the cat. *J. Physiol.* 389, 729–756.

during voluntary isometric contractions. *J. Physiol.* 230, 359–370.

in intrinsic excitability of human spinal motoneurones produced by natural activity. *J. Neurophysiol.* 108, 2473–2480.

human thenar motor units. *Muscle Nerve* 25, 77–82.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 October 2012; accepted: 06 April 2013; published online: 24 April 2013.*

*Citation: McNeil CJ, Butler JE, Taylor JL and Gandevia SC (2013) Testing the excitability of human motoneurons. Front. Hum. Neurosci. 7:152. doi: 10.3389/fnhum.2013.00152*

*Copyright © 2013 McNeil, Butler, Taylor and Gandevia. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## IH activity is increased in populations of slow versus fast motor axons of the rat

#### *Chad Lorenz1 and Kelvin E. Jones1,2 \**

*<sup>1</sup> Faculty of Physical Education and Recreation, University of Alberta, Edmonton, AB, Canada*

*<sup>2</sup> Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada*

#### *Edited by:*

*Annie Schmied, National Center for Scientific Research, France*

#### *Reviewed by:*

*Penelope A. McNulty, Neuroscience Research Australia, Australia Karl Ng, University of Sydney, Australia*

#### *\*Correspondence:*

*Kelvin E. Jones, Faculty of Physical Education and Recreation, University of Alberta, Edmonton, AB T6G 2H9, Canada*

*e-mail<EMAIL_ADDRESS>
Much is known about the electrophysiological variation in motoneuron somata across different motor units. However, comparatively less is known about electrophysiological variation in motor axons and how this could impact function or electrodiagnosis in healthy or diseased states. We performed nerve excitability testing on two groups of motor axons in Sprague–Dawley rats that are known to differ significantly in their chronic daily activity patterns and in the relative proportion of motor unit types: one group innervating the soleus ("slow motor axons") and the other group innervating the tibialis anterior ("fast motor axons") muscles.We found that slow motor axons have significantly larger accommodation compared to fast motor axons upon application of a 100 ms hyperpolarizing conditioning stimulus that is 40% of axon threshold (*Z* = 3.24, *p* = 0.001) or 20% of axon threshold (*Z* = 2.67, *p* = 0.008). Slow motor axons had larger accommodation to hyperpolarizing currents in the current-threshold measurement (−80% *Z* = 3.07, *p* = 0.002; −90% *Z* = 2.98, *p* = 0.003). In addition, we found that slow motor axons have a significantly smaller rheobase than fast motor axons (*Z* = −1.99, *p* = 0.047) accompanied by a lower threshold in stimulus-response curves. The results provide evidence that slow motor axons have greater activity of the hyperpolarization-activated inwardly rectifying cation conductance (IH) than fast motor axons. It is possible that this difference between fast and slow axons is caused by an adaptation to their chronic differences in daily activity patterns, and that this adaptation might have a functional effect on the motor unit. Moreover, these findings indicate that slow and fast motor axons may react differently to pathological conditions.

**Keywords: axon physiology, ion channels, hyperpolarization-activated inwardly rectifying cation conductance, nerve excitability test**

#### **INTRODUCTION**

Many studies have demonstrated that changes in chronic daily activity patterns alter the electrophysiological properties of cat and rodent motor neurons measured from the soma: after exercise (Beaumont and Gardiner, 2002, 2003; Gardiner et al., 2005), chronic stimulation (Munson et al., 1997), and hindlimb unweighting or spinal transections (Hochman and McCrea, 1994; Cormery et al., 2005). Muscle contractile properties also change in response to different activity levels like chronic stimulation and exercise (Kernell et al., 1987; Westgaard and Lomo, 1988; Gordon et al., 1997) or decreased activity levels through spinal transection (Cope et al., 1986; Munson et al., 1986). However, there is comparatively little known about the association between the electrophysiological properties of motor axons and motor unit type, or whether the axons change in response to altered chronic activity levels that induce plasticity of motor unit phenotype. The primary exception is conduction velocity (CV), which is altered in response to chronic changes in daily activity patterns of the motor units (Carp and Wolpaw, 1994; Munson et al., 1997; Beaumont and Gardiner, 2002). However, it has been acknowledgd for some time that conventional nerve CV provides limited physiological insight and additional measures from nerve excitability studies

are warranted (Krarup and Moldovan, 2009; Kiernan and Lin, 2012).

Nerve excitability studies in humans have demonstrated that electrophysiology is different between axons innervating different muscles (Kuwabara et al., 2000, 2001; Krishnan et al., 2004; Bae et al., 2009; Jankelowitz and Burke, 2009) and between motor axons innervating the same muscle but activated at different percutaneous stimulation thresholds (Trevillion et al., 2010). Similar studies in rodents suggested that motor axons activated at different stimulation thresholds vary moderately in measures of nerve excitability (Mori et al., 2010; Nodera and Rutkove, 2012). These studies suggest that motor axon conductances such as hyperpolarization-activated inwardly rectifying cation conductance (IH) and slow potassium conductance (IK s) may vary across muscles and axons of different threshold. However, none of these studies have examined populations of axons from muscles with distinct distributions of motor unit types. The previous research suggests that the major differences in motor axons are a result of differences in conductances rather than simply a consequence of different axon diameters.

The purpose of this study is to compare the electrophysiology of axons innervating the soleus muscle (SOL) to those innervating the tibialis anterior (TA) muscle in the rat. SOL axons were considered to represent a population of *slow-* and TA axons *fast-* motor axons. About 94% of rat TA motor units are classified as a fast (fast fatigable or fast fatigue-resistant) motor unit type while 6% are slow, based on a modified version of Burke's criteria originally used to distinguish motor units in the cat hindlimb (Totosy de Zepetnek et al., 1992). Note the motor unit distribution in the human TA has a much larger contribution from slow motor units (Johnson et al., 1973). On the other hand, about 80% of the motor units in rat SOL are classified as slow motor unit type while 20% are the fast motor unit type (Gillespie et al., 1987). Rat TA and SOL motor units have dramatically different activity patterns in locomotion (Gorassini et al., 2000) and presumably much different overall daily activity patterns (e.g., Hennig and Lomo, 1985). Thus, by using TA and SOL axons in this study, we analyzed axons of different motor unit types as well as axons with different chronic daily firing patterns.

#### **MATERIALS AND METHODS**

#### **SURGERY AND RECORDINGS**

A total of 14 female Sprague–Dawley rats, weighing 280 ± 50 g (mean ± SD), were used in this study. These weights in female Sprague–Dawley rats correspond to an age of approximately 90 days, which represents young but sexually mature females (Yang et al., 2000; George and Bostock, 2007). Rats were housed in pairs in a 12:12 h light–dark cycle, environmentally controlled (22– 24◦C, 40–70% humidity) room. Water and rat chow (Lab Diet 5001, PMI Nutrition, Brentwood, MO, USA) were provided *ad libitum*. All animal studies were conducted in accordance with the Canadian Council on Animal Care Guidelines and Policies with approval from the Animal Care and Use Committee: Health Sciences for the University of Alberta.

Anesthesia was induced by intraperitoneal injection of a mixed dose of 60 mg/kg ketamine and 7.2 mg/kg xylazine (KX) with additional KX dosages to maintain a surgical plane of anesthesia for the duration of the experiment. A rectal thermometer monitored internal body temperature, which was maintained between 34.5 and 38.5◦C by a heating lamp. Upon completion of all experiments, animals were euthanized by an overdose of KX followed by cervical dislocation.

An electrical clipper was used to initially remove hair over the lower back and legs followed by a depilatory cream to remove the remaining hair on the lower back and hip areas. Percutaneous electrical stimulation was delivered via Ag/AgCl 3M Red Dot electrodes (10 mm diameter), with the active electrode placed over the sciatic notch and the return electrode placed over the lumbar vertebrae at the midline of the back (**Figure 1A**). Electromyographic (EMG) recordings were made using Teflon-coated stainless steel monofilament wire (CoonerWire 765–40; 40G) with approximately 3 mm of Teflon removed from the end of the wire. The wire was threaded through a hypodermic needle (TA 26G, SOL 27G) with ∼2 mm extending from the tip and twisted to form a hook. To record a bipolar intramuscular compound muscle action potential (CMAP) signal in TA the first needle was inserted near the motor endplate on the proximal 1/3 of the muscle, and a second needle was inserted about 2.5 mm distally. Insertion of the hypodermic needles in SOL was similar except they were

placed about 5 mm apart because of the orientation of the motor endplate and muscle fibers in SOL. After the stainless steel recording electrodes were securely placed in each muscle, the needles were removed. To minimize EMG from other muscles and contamination of the target CMAP during the threshold tracking procedure, extensor digitorum longus, flexor digitorum longus, medial gastrocnemius, and plantaris were denervated. The distal 3/5 of lateral gastrocnemius (LG) was excised, rather than denervated, because SOL axons are intertwined with LG axons in a shared nerve branch that is not easily separated in the rat (**Figure 1B**).

Compound muscle action potentials were amplified (to cover one-half to two-thirds of the A/D range), filtered (10–1000 Hz) and digitized by a 12-bit A/D board (National Instruments DAQ-6062e, Austin, TX, USA) at a rate of 10 kHz. Noise introduced from nearby power sources was removed in real time using a Hum Bug 50/60 Hz Noise Eliminator (Quest Scientific Instruments, North Vancouver, BC, Canada). In cases of excessive levels of noise which occurred in six rats, a 60 Hz notch filter was also implemented. The time required from anesthetic induction to complete all measures in an animal was typically 1 h. In five animals the electrodes were repositioned and the tests repeated as a result of signal contamination. The time required in these cases was approximately 1.5 h. Arterial blood gases and acid base status were not monitored but are well maintained at rest in this preparation over this time period (Jendzjowsky and DeLorey, 2013).

#### **NERVE EXCITABILITY TESTING**

Multiple excitability measures of TA and SOL axons were made from the right leg. Stimuli were delivered using QTRAC software (Institute of Neurology, Queen Square, London, UK) and an isolated bipolar constant current stimulator (Digitimer DS5, Digitimer Ltd., Welwyn Garden City, Hertfordshire, UK). The QTRAC protocol "TRONDCMW" consists of five nerve excitability indices: threshold electrotonus (TE), current-threshold (I/V), recovery cycle (RC), rheobase, and strength-duration time constant (SDTC). The primary outcome measure for each of these indices is the change in the amplitude of the test stimulus required to produce a target CMAP: in this case 40% of the maximum with a tolerance of ±7.5%. The duration of the test stimulus was 1.0 ms except during rheobase and SDTC in which the duration varies between 0.2 and 1.0 ms. Krishnan et al. (2009) has a more detailed description of this validated methodology.

For TE, a 100 ms sub-threshold conditioning stimulus with an amplitude expressed as a percentage (±40 and ±20%) of the test stimulus is applied and the change in test stimulus amplitude is measured at multiple delays relative to the start of the conditioning stimulus. The I/V is a similar test with a 200 ms sub-threshold conditioning stimulus and the change in test stimulus amplitude is measured at the end of the conditioning stimulus. The amplitude of the conditioning stimulus for I/V measurements varies from +50 to −100% of the unconditioned test stimulus. In RC, a supramaximal conditioning stimulus is applied to the nerve, and the test stimulus is applied at delays ranging from 2 to 200 ms. Rheobase and SDTC are determined via linear regression of the charge-duration data obtained through five test stimuli that range in duration from 0.2 to 1.0 ms.

#### **ANALYSIS**

All statistical analysis was done in SPSS version 21. Normality was assessed by the Shapiro–Wilk test and it was found that most data groups (groups within each of the five nerve excitability indices) *violated* the normality assumption at an alpha level of 0.05. Specifically, the hypothesis of a normal distribution was rejected for all data groups except TE +20% and depolarizing I/V measures. Therefore the non-parametricWilcoxon signed-rank test was used for paired comparison of TA and SOL axons within each individual rat. For comparisons involving TE, paired comparison of TA and SOL axons were made at three delays for each of the four conditioning stimulus levels, i.e., ±40 and ±20%. For comparisons involving I/V, there were five data groups analyzed from I/V +50 to +10% and 10 groups analyzed from I/V 10 to −100%, all spaced by increments of 10%. In RC, all 18 delays ranging between 2.0 and 200 ms delay were analyzed. Finally, rheobase and SDTC were also used to compare TA and SOL axons. In order to control for type I statistical errors, we divided the alpha level

(0.05) by the number of data groups within each respective nerve excitability measure. Therefore, the adjusted alpha level (αa) for determining statistical significance was 0.017 in depolarizing and hyperpolarizing TE, 0.01 in depolarizing I/V, 0.005 in hyperpolarizing I/V, 0.006 in hyperpolarizing I/V Slope, 0.003 in RC, and 0.05 in rheobase and SDTC. The whiskers in each box-and-whisker plot represent 5 and 95% confidence intervals, while the box represents the interquartile range and the line the median. Line plots include the median value and error bars are the 95% confidence interval.

#### **RESULTS**

#### **THRESHOLD ELECTROTONUS**

Previous studies using nerve excitability testing have indicated that particular testing delays within the TE measurement are of greater interest since these are where properties diverge (Schwarz et al., 2006; George and Bostock, 2007; Sittl et al., 2011). These delays are 20–30, 100–109, and 120–150 ms and constitute the planned contrasts for statistical analysis (**Figure 2A**, gray areas). We averaged all the data within each of these delay ranges and used these values for comparisons between TA and SOL axons. **Figure 2A** displays the averaged data from TA and SOL axons for depolarizing TE +40% and hyperpolarizing TE −40%.

The initial fast phase, which is proportional to the applied current, was the same for TA and SOL axons for all depolarizing and hyperpolarizing conditioning currents. During depolarizing TE +40% there was no statistical difference between TA and SOL axons. In hyperpolarizing TE −40%, TA axons had a significantly greater threshold increase than SOL axons at 20–30 ms (*Z* = 3.24, *p* = 0.001) and 100–109 ms (*Z* = 3.24, *p* = 0.001), and a significantly smaller threshold decrease at 120–150 ms (*Z* = −2.61, *p* = 0.009; **Figure 2B**). For the TE −20% condition at 100– 109 ms, TA axons had a significantly greater threshold increase than SOL axons (*Z* = 2.67, *p* = 0.008; **Figure 2C**). The data for these measures are given in **Table 1**.

#### **CURRENT-THRESHOLD**

There was no significant difference between TA and SOL axons at any of the five depolarizing conditioning strengths. At hyperpolarizing conditioning strengths, however, we found that TA axons had significantly greater threshold increases at I/V −80% (*Z* = 3.07, *p* = 0.002) and −90% (*Z* = 2.98, *p* = 0.003; **Figure 3**).

The slopes of the TA and SOL I/V curves were calculated at hyperpolarizing conditioning strengths, and the slope of the TA axon line was found to be significantly smaller than the SOL axon line at I/V −40% (*Z* = 2.79, *p* = 0.045) and −50% (*Z* = 3.10, *p* = 0.018). The values for these I/V measurements are given in **Table 1**.

#### **RECOVERY CYCLE**

There were no significant differences between TA and SOL axons in RC (**Figure 4**; **Table 1**) using the adjusted alpha value of 0.003 for multiple comparisons. Comparison at delays of 2–5 ms showed that TA axons had smaller increases in threshold compared to SOL axons (*p*-values less than 0.05), but these differences did not meet criteria for rejection of the null-hypothesis given the multiple comparisons.

**FIGURE 2 |Threshold electrotonus. (A)** Average values for depolarizing and hyperpolarizing conditioning pulses with 40% amplitude. Planned statistical comparisons of TA and SOL axons were done at delay ranges illustrated with a gray background. **(B)** Percent change in threshold for the hyperpolarizing 40% threshold electrotonus (TE) condition. The whiskers in each box-and-whisker plot represent 5 and 95% confidence intervals, while the box represents the interquartile range and the midline the median. TA data in

hatched boxes, SOL in gray boxes. Wilcoxon signed-rank tests, which are paired comparisons, show a statistically significant increase in median "Threshold Decrease (%)" for SOL compared to TA axons at all delays. **(C)** Percent change in threshold for the hyperpolarizing 20% TE condition. There is a statistically significant increase in median threshold change for SOL compared to TA axons, only at the end of the hyperpolarizing condition pulse, i.e., 100–109 ms. ∗*p* < 0.017.

#### **STIMULUS-RESPONSE CURVES, RHEOBASE, AND STRENGTH-DURATION TIME CONSTANT**

The stimulus-response curves showed a rightward shift for the TA axons indicating a higher threshold (**Figure 5A**). The normalized stimulus-response curve for TA was less steep compared to that for the SOL axons (**Figure 5B**). Paired comparisons found that TA axon rheobase was modestly but significantly larger than SOL axon rheobase (*Z* = 1.99, *p* = 0.047; **Figure 5C**). The higher rheobase for TA axons is consistent with the rightward shift of the stimulusresponse curve. The SDTC was not different between the two axon groups (*Z* = 1.36, *p* = 0.17; **Figure 5D**).

#### **DISCUSSION**

The present findings show that many measures of nerve excitability are indistinguishable for slow soleus and fast TA motor axons in the rat. The exception to this general finding is for measures of accommodation to prolonged hyperpolarization during the TE and I/V tests. The most parsimonious explanation for the differences is that voltage-gated inwardly rectifying current IH is greater in slow compared to fast axons. However, some of the differences can not be explained by differences in IH: stimulus-response curves and rheobase, as well as differences in −40% TE at the early delay of 20–30 ms.

**Table 1 | Mean values and 95% confidence intervals for the dependent variables generated by nerve excitability testing (NET) of** *fast* **TA versus** *slow* **SOL motor axons.**

*The alpha criterion was adjusted (*αa*) for multiple comparisons for each of the main outcome measures. TE* = *0.017, depolarizing I/V* = *0.01, hyperpolarizing I/V* = *0.005, I/V slope* = *0.006, RC* = *0.003, and 0.05 for rheobase and SDTC.* ∗ *indicates p-values less than these adjusted alpha.*

The most direct evidence implicating the channel responsible for IH, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, in TE and I/V nerve excitability measures comes from direct *in vitro* voltage recording of rat spinal root myelinated axons (Baker et al., 1987). HCN channels are blocked by cesium (Biel et al., 2009) and Baker et al. (1987) showed that the late accommodation in measures similar to TE and I/V was antagonized by cesium. Yang et al. (2000) used cesium chloride to decrease accommodation to hyperpolarizing TE measures using an *in vivo* rat, tail motor axon preparation similar to the current study. For TE −20 and −40% conditioning pulses the threshold reduction at delays of 100–109 ms was greater after cesium administration. Thus reducing IH in mixed tail motor axons generates responses that are similar to the data from fast TA motor axons reported here

**FIGURE 3 | Conditioning current versus threshold.** For conditioning strengths of −80 and −90% there is a significantly smaller decrease in threshold for SOL compared to TA axons indicative of stronger depolarization current activated by the hyperpolarization for SOL axons. The difference between percent threshold decrease of SOL and TA axons at −80% was 22.3, 25.8 at −90% and 29.8 at a conditioning strength of −100%. Increased variance at −100% likely contributed to the failure to meet the threshold alpha of 0.006 to reject the null hypothesis.

**FIGURE 4 |The recovery cycle in the rat TA and SOL motor axons did not show a period of superexcitability followed by subexcitability, just a slowly decaying relative refractory period.** A Wilcoxon signed-rank test did not find any significant differences between TA and SOL axons at any of the 18 delays. It should be noted that the large number of delays (18) that were compared created a very small adjusted alpha level, 0.003. Therefore there is an increased risk of type 2 error.

(**Figure 2**). This supports our inference that fast motor axons have weaker IH activity compared to slow motor axons.

This is not the first study to suggest that IH may vary across a population of motor axons, but it is the first to test two clearly distinct populations. A previous study investigated motor axons in

human median nerve to conclude that the lowest threshold axons had different electrophysiological properties compared to higher threshold axons (Trevillion et al., 2010). The best explanation for the unique properties of low-threshold motor axons was that they had a greater level of IH activity, and this inference was based on computer modeling. The authors used *F*-wave latency to characterize ten single motor units (45% of their total sample) as having a faster CV and therefore larger diameter compared to the average motor axon in the population responses. Their conclusion was that axons of a larger diameter and faster CV had greater IH activity. This is the opposite of the findings in the present study that found greater IH activity in the slower soleus motor axons compared to fast TA motor axons. It is not clear at this time why there is a clear discrepancy in the two findings other than the obvious species difference and use of anesthetic.

There are four isoforms of the HCN channel, HCN1–HCN4, all of which allow for passage of a mixed inward cationic current of both K+ and Na+ ions. HCN channels are found in all three major neuron compartments: the soma, dendritic tree, and the axon proper (Baker et al., 1987; Takigawa et al., 1998; Robinson and Siegelbaum, 2003; Biel et al., 2009). In addition to being voltage dependent, HCN channels have been found to be influenced by acidic lipids (i.e., PIP2), protons, extracellular K+ concentration, and cytosolic proteins, and usually most significantly by cAMP (Biel et al., 2009). HCN1 has the fastest time constant of activation which can range anywhere from 30 to 200 ms at −140 to −95 mV. HCN2 is the next fastest isoform with the time constant of activation ranging from 150 ms to 1 s (Biel et al., 2009). Two recent nerve excitability studies comparing nerve excitability properties of motor and sensory axons have discussed the hypothesis that differences in IH between these two axon groups are due to sensory axons having either greater expression of faster HCN isoforms and/or a different availability of HCN ligands that renders greater IH in sensory compared to motor axons (Howells et al., 2012; Nodera and Rutkove, 2012). A higher density of channels on slow axons, the expression of different isoforms, or cAMP dependent phosphorylation status generating a difference in half-activation

voltages could explain the increased IH in slow motor axons in the present study. There was no *a priori* hypothesis that IH current would dominate the difference between slow and fast axons. Future studies should use a modified nerve excitability testing protocol with additional stronger and more prolonged hyperpolarizing conditioning stimuli to further characterize the difference in IH between fast and slow axons (Tomlinson et al., 2010).

Thus, it appears IH is a plastic ionic conductance that varies according to the type of motor axon as well as between motor and sensory axons. As the type of activity patterns are different between most motor and sensory axons, as well as for lower and higher threshold axons, this points to the possibility that IH expression depends on impulse activity patterns. This is the interpretation given for the finding of reduced IH in human motor axons on the affected side for individuals experiencing hemiparesis subsequent to stroke (Jankelowitz et al., 2007). In these individuals the motor axons on the affected side are expected to have a chronic reduction in impulse activity, if spasticity is not excessive. The reduced chronic activity leads to an adaptive change in IH that is consistent with our finding of less accommodation to hyperpolarization in the rat TA motor axons. Axons innervating the rat TA are mostly from fast motor units that fire less often but at higher frequencies than the axons of SOL, which are mostly from slow motor units (Gillespie et al., 1987; Totosy de Zepetnek et al., 1992; Gorassini et al., 2000). Activity-dependent hyperpolarization of axons is known to correlate positively with firing frequency as well as the number of impulses in a train (Erlanger and Gasser, 1973; Raymond, 1979; Morita et al., 1993; Kiernan et al., 2004), although the magnitude of hyperpolarization saturates at frequencies around 20–50 Hz in lizards (Morita et al., 1993) and possibly at 20–30 Hz in humans (Kiernan et al., 2004). Although the relative importance of firing frequency and impulse load in creating activity-dependent hyperpolarization in rat TA and SOL axons is unclear, (Raymond, 1979) has shown that in the frog, average firing frequencies as low as 1.25 Hz can cause axon threshold depression and therefore probably activity dependent hyperpolarization (see Figure 9 in Raymond, 1979). Bursts in rat SOL motor units fire at an average frequency of 20 Hz for roughly 30% of the day (Hennig and Lomo, 1985), and so it seems likely that SOL axons would generally experience a greater tendency toward activity-dependent hyperpolarization than TA axons due to greater activation by the Na+–K+ pump. It is possible that greater IH expression in SOL axons offsets a tendency for their hyperpolarization caused by higher levels of activity.

While many of the differences in nerve excitability indices are consistent with the interpretation that IH is greater in SOL axons, this mechanism may not explain differences in the stimulusresponse curves, rheobase and early differences in hyperpolarizing TE. The finding that TA axons have a greater rheobase and have a rightward shift in the stimulus-response curve indicates that stronger stimuli were required to activate these axons. The magnitude of the differences was small and should be considered when assessing the importance of this finding. For example, rheobase differed on average by 0.06 mA that was much smaller than the SDs for this measure, which were 0.20 and 0.34 mA for TA and

SOL axons respectively. Typically differences in rheobase have not been interpreted as resulting from IH. Similarly, the rate of activation of HCN channels is typically considered too slow to contribute to differences in hyperpolarizing TE at the 20–30 ms delay (Tomlinson et al., 2010). It is important to emphasize that the difference reported in **Figure 2B** was not a result of different extents of the fast change in threshold at the onset of the conditioning stimuli. The typical delay for overt expression of the effects of IH is closer to 100 ms in human studies but can be much earlier if the membrane potential is depolarized (Bostock et al., 1998). There is some evidence from nerve excitability studies in mice that axons in the sciatic nerve may appear to be depolarized, even if there is no reason to suppose this to be the case (Boerio et al., 2009, 2010). In any case, it can reasonably be argued that if there is membrane depolarization, it should affect the TA and SOL axons equally and facilitate the measurement of greater IH in SOL axons at earlier delays. Alternatively, differences in tissue impedance between rat and human experiments may facilitate earlier onset of IH in rat experiments.

In conclusion, while for many nerve excitability measures we were not able to distinguish between slow and fast motor axons, they were significantly different during strong 100–200 ms hyperpolarizing conditioning stimuli. The axon threshold in these conditions is strongly affected by hyperpolarization-activated current IH and the findings suggest a greater level of IH in slow motor axons. This finding has implications for the use of nerve excitability testing in neurodegenerative conditions like amyotrophic lateral sclerosis (ALS). In the SOD1-G93A mouse model of ALS there is a clear motor unit type dependent vulnerability with fast-fatiguing motor units being most susceptible (Frey et al., 2000; Pun et al., 2006; Hegedus et al., 2008; Deforges et al., 2009; Kanning et al., 2010). As the vulnerable motor axons become denervated early in the disease the population of axons generating the nerve excitability measures will change. Longitudinal changes in nerve excitability measures (e.g., Kanai et al., 2006) may be confounded by the change in population of heterogeneous motor axons that contribute to the measure.

#### **AUTHOR CONTRIBUTIONS**

Chad Lorenz contributed to the acquisition, analysis and interpretation of the data, drafted the initial version of the manuscript, derived from a Master's thesis, and gave final approval for the submitted publication. Kelvin E. Jones conceived the study and contributed to acquisition, analysis, and interpretation of the data. The final manuscript was revised for publication by Kelvin E. Jones.

#### **ACKNOWLEDGMENTS**

This work would not have been possible without the expert technical assistance of Neil Tyreman. The research was supported by a Canadian Institutes of Health Research (CIHR) grant from the Neuromuscular Research Partnership (NRP) that includes support from Muscular Dystrophy Canada and the ALS Society of Canada.

#### **REFERENCES**

Bae, J. S., Sawai, S., Misawa, S., Kanai, K., Isose, S., and Kuwabara, S. (2009). Differences in excitability properties of FDI and ADM motor axons. *Muscle Nerve* 39, 350–354. doi: 10.1002/mus.21107

in the rat node of ranvier. *J. Physiol.* 573, 17–34. doi: 10.1113/jphysiol.2006. 106815

Yang, Q., Kaji, R., Hirota, N., Kojima, Y., Takagi, T., Kohara, N., et al. (2000). Effect of maturation on nerve excitability in an experimental model of threshold electrotonus. *Muscle Nerve* 23, 498–506. doi: 10.1002/(SICI)1097- 4598(200004)23:4<498::AID-MUS7>3.0.CO;2-U

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 June 2014; paper pending published: 06 August 2014; accepted: 10 September 2014; published online: 25 September 2014.*

*Citation: Lorenz C and Jones KE (2014) IH activity is increased in populations of slow versus fast motor axons of the rat. Front. Hum. Neurosci. 8:766. doi: 10.3389/fnhum.2014.00766*

*This article was submitted to the journal Frontiers in Human Neuroscience.*

*Copyright © 2014 Lorenz and Jones. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Asynchronous recruitment of low-threshold motor units during repetitive, low-current stimulation of the human tibial nerve

#### **Jesse C. Dean<sup>1</sup> , Joanna M. Clair-Auger <sup>2</sup> , Olle Lagerquist <sup>3</sup> and David F. Collins <sup>2</sup>\***

<sup>1</sup> Division of Physical Therapy, Medical University of South Carolina, Charleston, South Carolina, USA

<sup>2</sup> Faculty of Physical Education and Recreation and Centre for Neuroscience, University of Alberta, Edmonton, AB, Canada

<sup>3</sup> Northern Alberta Institute of Technology, Edmonton, AB, Canada

#### **Edited by:**

Parveen N. S. Bawa, Simon Fraser University, Canada

#### **Reviewed by:**

Kevin Power, Memorial University, Canada Stephan Riek, The University of Queensland, Australia

#### **\*Correspondence:**

David F. Collins, Faculty of Physical Education and Recreation and Centre for Neuroscience, University of Alberta, E-488 Van Vliet Centre, Edmonton, AB, T6G 2H9 Canada e-mail<EMAIL_ADDRESS>
Motoneurons receive a barrage of inputs from descending and reflex pathways. Much of our understanding about how these inputs are transformed into motor output in humans has come from recordings of single motor units during voluntary contractions. This approach, however, is limited because the input is ill-defined. Herein, we quantify the discharge of soleus motor units in response to well-defined trains of afferent input delivered at physiologically-relevant frequencies. Constant frequency stimulation of the tibial nerve (10–100 Hz for 30 s), below threshold for eliciting M-waves or H-reflexes with a single pulse, recruited motor units in 7/9 subjects. All 25 motor units recruited during stimulation were also recruited during weak (<10% MVC) voluntary contractions. Higher frequencies recruited more units (n = 3/25 at 10 Hz; n = 25/25 at 100 Hz) at shorter latencies (19.4 ± 9.4 s at 10 Hz; 4.1 ± 4.0 s at 100 Hz) than lower frequencies. When a second unit was recruited, the discharge of the already active unit did not change, suggesting that recruitment was not due to increased synaptic drive. After recruitment, mean discharge rate during stimulation at 20 Hz (7.8 Hz) was lower than during 30 Hz (8.6 Hz) and 40 Hz (8.4 Hz) stimulation. Discharge was largely asynchronous from the stimulus pulses with "time-locked" discharge occurring at an H-reflex latency with only a 24% probability. Motor units continued to discharge after cessation of the stimulation in 89% of trials, although at a lower rate (5.8 Hz) than during the stimulation (7.9 Hz). This work supports the idea that the afferent volley evoked by repetitive stimulation recruits motor units through the integration of synaptic drive and intrinsic properties of motoneurons, resulting in "physiological" recruitment which adheres to Henneman's size principle and results in relatively low discharge rates and asynchronous firing.

**Keywords: motoneuron, electrical stimulation, reflex, sensorimotor integration, motor unit**

#### **INTRODUCTION**

Understanding how motoneurons transform synaptic input into motor output is fundamental to understanding their role in the neural control of human movement. During voluntary contractions, motoneurons receive synaptic inputs from descending and reflex pathways. The present thinking is that the currents that drive motoneuron discharge come from synaptic inputs, intrinsic properties of the neurons themselves (e.g., persistent inward currents; PICs) and metabotropic mechanisms that regulate the strength of these currents over a wide range. These ideas about motoneuron discharge (see Heckman and Enoka, 2012 for review) are based on recordings from motoneurons in reduced animal models (Schwindt and Crill, 1980; Bennett et al., 1998b; Heckman and Lee, 2001), from motor units in humans (Kiehn and Eken, 1997; Gorassini et al., 1998, 2002a) and from computational models (Elbasiouny et al., 2006; Powers et al., 2012). A limitation of studying how motoneurons transform synaptic input into motor output during voluntary contractions in humans is that the temporal characteristics of the synaptic input are inherently ill-defined. One way to circumvent this problem is to study motor unit discharge in response to trains of electrically-evoked afferent impulses. In this way, the temporal characteristics of the synaptic drive are relatively well-defined and the relationship between synaptic drive and motor output can be quantified (Kudina, 1988; Jones and Bawa, 1995; Bawa and Chalmers, 2008; Binbo˘ga et al., 2011). The purpose of the present experiments was to characterize the recruitment and ongoing discharge of human motoneurons when they receive trains of afferent impulses over a range of physiologically relevant frequencies.

Presently, we deliver trains of impulses to human motoneurons by stimulating afferents in the tibial nerve at different frequencies and measure the output by recording the discharge of soleus motor units. The afferent volley evoked by a single suprathreshold pulse delivered to the tibial nerve comprises activity in axons from muscle spindles, Golgi tendon organs and cutaneous receptors (Burke et al., 1983) and traverses mono- and polysynaptic pathways to motoneurons (Burke et al., 1984). The first impulses reach the motor pool in ∼15 ms, with impulses traveling along slower axons and/or through polysynaptic pathways arriving ∼6– 10 ms later (Burke et al., 1984). Although the effects of the afferent volley on motoneurons can be both excitatory and inhibitory (Burke et al., 1983; Marchand-Pauvert et al., 2002; Binbo˘ga et al., 2011), the net result is a relatively synchronous discharge of the motor pool known as an H-reflex (Hoffmann, 1918), which is primarily due to inputs from Ia afferents acting through predominantly monosynaptic pathways (Pierrot-Deseilligny and Mazevet, 2000). During low-intensity repetitive stimulation of the tibial nerve, motor unit activity can develop gradually and has been qualitatively reported to be asynchronous from the stimulation pulses (Lang and Vallbo, 1967; Burke and Schiller, 1976; Collins et al., 2001). Similar contractions develop when vibration is applied over a tendon or muscle belly, a phenomenon known as the tonic vibration reflex (TVR; De Gail et al., 1966; Hagbarth and Eklund, 1966; Burke and Schiller, 1976). In contrast to during electrical stimulation, however, motor unit activity during the TVR has been reported to be phase-locked to the mechanical stimulus (Burke and Schiller, 1976). More recently, we have shown that during repetitive stimulation of the tibial nerve motor unit discharge can be both synchronous with each stimulus pulse, as an H-reflex (Klakowicz et al., 2006; Bergquist et al., 2011; Clair et al., 2011), and "asynchronous" from each stimulus pulse (Collins et al., 2001; Bergquist et al., 2011). The current study represents a quantitative analysis of how such trains of afferent input are transformed into motor output.

We delivered electrical stimulation to the tibial nerve at a low current, below the threshold at which a single pulse elicited a measurable soleus M-wave, H-reflex or ankle extensor torque. Stimulating at such a low current minimized the number of motor units recruited during repetitive stimulation and thus reduced the chances of more than one motor unit discharging simultaneously, at an H-reflex latency, enabling us to more easily quantify motor unit discharge patterns. Low current stimulation also avoided participant discomfort and thus the potential of participants "tensing-up" and/or producing descending commands that contribute to motor unit recruitment. Our working hypothesis is that the recruitment and ongoing discharge of motor units during repetitive electrical stimulation in humans reflects the integration of currents that arise from synaptic drive and intrinsic properties of the neurons themselves, resulting in motor unit discharge that can be either "time-locked" to each stimulus pulse (i.e., H-reflexes) or "asynchronous" from the stimulus pulses. We predicted that low-current electrical stimulation will recruit motor units that are recruited during weak voluntary contractions (Sypert and Munson, 1981), consistent with the synaptic source of recruitment which follows Henneman's size principle (Henneman et al., 1965; Calancie and Bawa, 1984), and that recruitment will occur over relatively long time periods (on the order of seconds), consistent with the amplification of synaptic inputs by PICs in motoneurons (Spielmann et al., 1993; Bennett et al., 1998a; Heckman and Lee, 2001; Gorassini et al., 2002b). We also predicted that recruitment will occur with no measurable increase in synaptic drive, as quantified by a modified version of the paired motor unit technique. The results of the detailed analyses of motor unit discharge during repetitive stimulation described herein provide insight into how sensory input is transformed into motor output in humans which may prove to be useful for investigating pathophysiological aspects of sensorimotor integration after injury or disease.

#### **METHODS**

Experiments were conducted on nine healthy adult volunteers (7 male, 2 female) ranging in age from 22 to 44. All subjects provided written informed consent before participation in the study. The experiments were approved by the University of Alberta Health Research Ethics Board and were conducted in accordance with the Declaration of Helsinki. Low-current electrical stimulation was delivered over the tibial nerve while soleus motor unit activity and ankle extensor torque were recorded. Motor unit recruitment latencies and discharge patterns were compared for a range of stimulation frequencies.

#### **EXPERIMENTAL SETUP**

To record isometric ankle extensor torque, subjects were seated on the chair of a Biodex dynamometer (System 3, Biodex Medical Systems, Inc, Shirley, NY, USA) with the right hip flexed to 110◦ , the right knee flexed to 90◦ , the right ankle at 90◦ , the right lateral malleolus aligned with the axis of the dynamometer, and the foot strapped to the Biodex footplate. The subject's trunk was at an approximate angle of 20◦ reclined from the vertical. Surface EMG was collected using self-adhesive electrodes (1" × 1", Disposable A10043 Gel Electrodes; Vermed Inc. Bells Falls, VT, USA) placed over the soleus.

Subjects completed 1–3 submaximal contractions of the ankle extensors to warm up prior to the collection of maximum isometric voluntary contraction (MVC) data. They were instructed to push down as if they were pressing a gas pedal, rapidly increase force to a maximum and hold this contraction for 5 s. Following the practice trials, each subject completed between 2 and 4 MVCs, separated by one minute of rest, until the MVC torques varied by less than 10% for two successive contractions. The MVC was quantified as the maximum torque achieved in a single trial during the time period beginning 1 s after the start of the contraction.

After the MVCs were completed, fine wires (0.002 inch diameter, stainless steel; A-M Systems Inc. Carlsborg, WA, USA) were inserted into the soleus muscle belly using a 23-gauge needle to record the activity of single motor units. After insertion, subjects held a weak voluntary contraction (<10% MVC) while the fine wires were slowly retracted until a clear individual motor unit was detected. All EMG data were band-pass filtered between 30 and 5000 Hz. All data were sampled at 10,000 Hz and stored for subsequent analysis.

#### **ELECTRICAL STIMULATION**

Surface electrodes (1" × 1", Disposable A10043 Gel Electrodes; Vermed Inc. Bells Falls, VT, USA) were placed behind the knee in the popliteal fossa to stimulate the tibial nerve. Rectangular electrical pulses (1 ms duration) were delivered using a Digitimer constant current stimulator (DS7A, Welwyn Garden City, Hertfordshire, England) under computer control. Before each trial, the stimulation current was varied in 0.05 mA increments to find the highest current at which single pulses were sub-threshold for both M-waves and H-reflexes in both the surface and fine wire motor unit EMG and did not produce any detectable ankle extensor torque. Subjects were instructed to remain relaxed during the stimulation, and none reported any discomfort.

Prior to the electrical stimulation trials, subjects performed a voluntary contraction in which they increased ankle extensor torque up to approximately 10% of their MVC, an estimate of the expected maximum torque during the electrical stimulation trials, and then decreased the contraction back to rest. Subjects were instructed to increase and decrease the strength of their contraction as slowly as possible. At the beginning of each electrical stimulation trial, subjects received 3 single pulses of stimulation separated by 5 s to confirm that the stimulation current was sub-threshold. If an M-wave, H-reflex, or torque response was present, the stimulation intensity was reduced and the trial was restarted. Five seconds after the last single pulse, a 30 s stimulation train was delivered at one of seven frequencies: 10, 20, 30, 40, 60, 80, or 100 Hz. The order of these frequencies was randomized for each subject. In many trials soleus EMG activity and ankle extensor torque remained after the stimulation was turned off, in which case subjects were instructed to "relax completely" (>1 s after the end of stimulation), which has been shown to terminate any involuntary sustained activity (Collins et al., 2002). After the seven electrically stimulated trials, one for each frequency, another voluntary contraction with slowly increasing and decreasing torque was performed, with torque increased to the maximum torque level produced during the electrically stimulated trials. The trials for voluntary contractions were performed to determine the torque level at which motor units were first recruited voluntarily and to ensure that the same motor units were recorded during the different electrical stimulation trials. Two minutes of rest separated each trial.

Upon completion of these trials, the fine wire electrodes were slowly retracted while the subjects maintained a weak voluntary contraction. The electrodes were moved until the original motor unit was no longer detectable and a new motor unit was identified. The procedure described in the paragraph above was then repeated. This series of steps was performed until either: (1) the fine wire electrode was pulled out of the muscle belly; (2) four hours had elapsed from the beginning of the experiment; or (3) the subject expressed discomfort from sitting in the same position for the duration of the experiment.

#### **MOTOR UNIT DETECTION**

Single motor unit data were analyzed using Spike 2 software (Cambridge Electronic Design Limited; Cambridge, UK). Individual motor units were discriminated using the template matching function of the software and validated by visual inspection. Discriminating individual motor units during electrical stimulation can be complicated by the simultaneous firing of multiple motor units at M-wave or H-reflex latencies, particularly when the stimulation is suprathreshold for M-waves and/or H-reflexes. At the low stimulation currents used for the majority of the trials in this study, motor unit discharge was often unrelated to the timing of each stimulation pulse. This, along with the relatively small number of motor units recruited by the lowcurrent stimulation, allowed easier discrimination of individual motor units.

In the present experiments, we compared the discharge patterns of motor units recruited by a range of stimulation frequencies, and ensured that we were analyzing the same motor unit during successive trials using *post-hoc* template matching. To confirm that the fine wire electrodes did not change location during the electrical stimulation trials, we compared the motor units recruited by a voluntary contraction before these trials to the motor units recruited voluntarily after the electrical stimulation trials. If the same motor units were not activated, the electrical stimulation data were not used.

#### **DATA ANALYSIS AND STATISTICS**

We were interested in the effect of different stimulation frequencies on the recruitment and firing patterns of an individual motor unit. Therefore, we performed detailed analysis only on motor units that were recruited by at least 3 stimulation frequencies; motor units had to be recruited with a stimulation frequency at or below 60 Hz. With this recruitment criterion, we analyzed data collected from a total of 25 distinct motor units.

To address the predictions derived from our working hypothesis, we quantified the following characteristics of motor unit discharge, which are described in more detail in the subsequent paragraphs: (1) ankle extensor torque at recruitment; (2) number of recruited motor units and recruitment latency; (3) temporal relationship between stimulation pulses and the discharge of single motor units; (4) instantaneous discharge rate after electrical stimulation; and (5) discharge rate at the time of recruitment of additional motor units. For each of the statistical tests, *post-hoc* tests were performed as appropriate when significance (*p* < 0.05) was found. *Post-hoc* Tukey comparisons were performed following ANOVAs, and *post-hoc* reduced Chi-square comparisons with a Bonferroni correction were performed following Chi-square tests. All descriptive statistics are reported as mean ± standard deviation.

#### **ANKLE EXTENSOR TORQUE AT RECRUITMENT**

We predicted that low-current stimulation would recruit low threshold motor units. To test this, we calculated the ankle extensor torque at the time of motor unit recruitment during both electrically stimulated and voluntary contractions, normalized by the subject's MVC ankle extensor torque. Motor unit recruitment was defined as the beginning of continuous motor unit firing. In some cases, motor units discharged once or twice, but then went silent again before beginning to fire continuously. Therefore, the beginning of continuous firing was calculated as the first motor unit discharge for which all subsequent interspike intervals were shorter than 600 ms. We used the criteria of 600 ms to identify "continuous" firing as was used previously by Gorassini et al. (2002b) based on the work of Matthews (1996) who identified 300 ms as the longest inter-spike interval for continuously firing soleus motor units. By using a criteria that was double this minimum value we were confident that we were not including in our analyses periods of isolated or sporadic motor unit activity.

#### **NUMBER OF MOTOR UNITS RECRUITED AND RECRUITMENT LATENCY**

Based on our working hypothesis, we expected higher stimulation frequencies to recruit more motor units at shorter latencies. To determine whether stimulation frequency had a significant effect on the number of motor units recruited (out of 25) we performed a Pearson Chi-square analysis. For each trial in which a motor unit was recruited by electrical stimulation, we calculated the recruitment latency as the time between the start of stimulation (i.e., the first stimulus pulse in the train) and the onset of continuous firing as described above. A repeated measures one-way ANOVA was performed to determine whether stimulation frequency had a significant effect on recruitment latency.

#### **TEMPORAL RELATIONSHIP BETWEEN STIMULATION PULSES AND SUBSEQUENT DISCHARGES**

To determine whether motor unit firing was synchronous or asynchronous from the stimulation pulses, we generated poststimulus time histograms (PSTHs; bin width = 1 ms) of the motor unit discharges following each stimulation pulse for all trials in which the stimulation rate was 10 or 20 Hz. At higher stimulation frequencies, the interval between stimulation pulses was too short (≤33.3 ms) to reliably detect motor units firing at an H-reflex latency (typically ∼35 ms). For all trials at 10 and 20 Hz, we calculated the mean and standard deviation of the number of motor unit discharges for each bin of the PSTH. We judged synchronous firing to occur when the number of motor unit discharges at a given latency exceeded the mean plus two times the standard deviation. For all trials, there was a single peak of synchronous firing that was 1–2 ms in duration and occurred between 35 and 44 ms after the stimulation pulse. These peaks were defined to be due to motor units firing at an H-reflex latency. Similar peaks at an M-wave or any other latency were never seen.

We predicted that motor units would fire at a rate within a narrow range, largely asynchronous from the stimulation pulses. The instantaneous discharge rate was calculated for all motor units recruited by 10–40 Hz electrical stimulation. At higher stimulation frequencies more stimulation artifacts were present per unit time and more motor units were recruited, making individual motor units more likely to become obscured. Peak discharge rate was defined as the instantaneous discharge rate values during the 5 s period with the highest average value. We performed repeated measures one-way ANOVAs to identify significant differences in the onset period and the peak discharge rate during 20, 30, and 40 Hz stimulation. These comparisons were performed for the nine motor units that were recruited at each of these stimulation frequencies.

Motor units that fired at an H-reflex latency after a stimulation pulse were assumed to fire as a result of the electrically-evoked afferent input. Therefore, we quantified the time between each stimulation pulse and the previous motor unit discharge, hereby termed "after firing period", and calculated the probability of a stimulation pulse delivered at this time causing a motor unit to fire at an H-reflex latency. We considered this probability an indicator of motor unit excitability, as a motor unit closer to its firing threshold would be more likely to fire in response to the electrically-evoked afferent volley.

#### **INSTANTANEOUS DISCHARGE RATE AFTER CESSATION OF ELECTRICAL STIMULATION**

If, as hypothesized, motor unit discharge was at least partially driven by activation of PICs, we would expect firing to be sustained beyond the end of stimulation. Sustained firing was deemed to be present if a motor unit continued to fire for at least one second after the end of electrical stimulation. To determine whether the electrically-evoked afferent volley influenced motor unit discharge rate, we performed a repeated measures one-way ANOVA to compare the discharge rate during the last second of stimulation to that during the first second of sustained firing after stimulation ended. This comparison was performed for the nine motor units that were recruited at stimulation frequencies of 20, 30, and 40 Hz.

#### **DISCHARGE AT THE TIME OF RECRUITMENT OF ADDITIONAL MOTOR UNITS**

Finally, we predicted that motor unit recruitment would occur without an increase in synaptic drive. We tested this using data from trials in which multiple motor units were recruited during the 30 s of electrical stimulation. In a modified version of the "paired motor unit recording" technique (Kiehn and Eken, 1997; Gorassini et al., 1998, 2002a,b), we monitored the discharge rate of the first recruited motor unit (the "control unit") at the time when an additional motor unit (the "test unit") was recruited. This approach relies on the assumption that there is a linear relationship between the synaptic drive received by the control and test motor units. If the discharge rate of the control unit was constant, this was taken as an indication that the synaptic drive to the motor pool was also approximately constant. To identify any changes in discharge rate, our surrogate measure of synaptic drive, we performed a repeated measures one-way ANOVA to determine whether the control unit instantaneous discharge rate changed significantly from the 1 s prior to the recruitment of the test unit to the 1 s period following this recruitment. We also tested whether discharge rates were affected by the afferent volley produced by the electrical stimulation by performing a repeated measures one-way ANOVA comparing discharge rate during the last second of stimulation and the first second of sustained firing for both control and test units. These comparisons were performed for the ten instances in which two distinct motor units were recruited by 10, 20, 30, or 40 Hz stimulation.

#### **RESULTS**

Electrical stimulation was delivered at a current below the threshold required for a single pulse to elicit either an M-wave or an H-reflex, or produce any ankle extensor torque (**Figure 1A**). When this stimulation, which ranged from 2.5–10 mA across participants, was delivered at a constant frequency (10–100 Hz), soleus motor unit activity and ankle extensor torque developed gradually in seven of the nine subjects and thus analyses were performed only on data from those seven subjects. **Figure 1** shows data from a single subject in whom stimulation at 60 Hz (panel B) resulted in the gradual development of torque (∼4% MVC) and the recruitment of 2 distinct motor units whose discharge was sustained after the stimulation ended. Data from 25 motor

units that fit our recruitment criterion (see Methods) were analyzed.

#### **ANKLE EXTENSOR TORQUE AT RECRUITMENT**

Each of the motor units recruited during low-current electrical stimulation was also recruited during a voluntary contraction of similar strength. An example of data recorded from a single subject is shown in **Figure 2**. All 25 motor units recruited during electrical stimulation were recruited with a relatively weak (<10% MVC) voluntary contraction.

#### **NUMBER OF MOTOR UNITS RECRUITED AND RECRUITMENT LATENCY**

During 30 s of constant frequency low-current stimulation, motor units did not respond to the first stimulation pulse, but rather were silent for a given latency before being recruited (**Figure 3A**), or were not recruited at all. Higher stimulation frequencies recruited significantly more motor units within the 30 s of stimulation (Chi-square, *p* < 0.05; **Figure 3B**). A motor unit recruited by a given stimulation frequency was always recruited by each of the higher stimulation frequencies. For example, the 3 motor units recruited at 10 Hz stimulation were also recruited at all of the other stimulation frequencies.

Recruitment latencies spanned almost the entire course of the electrical stimulation, ranging from 0.5 to 29.6 s (**Figure 3C**). Several of the motor units were not recruited for a relatively long time, as 22 of the motor units had a recruitment latency longer than 10 s in at least one trial, and 9 of the motor units had a recruitment latency longer than 20 s in at least one trial. Higher stimulation frequencies recruited motor units at significantly shorter latencies than low frequencies (ANOVA, *p* < 0.05).

This is illustrated in **Figure 3D**, in which the 25 motor units are grouped into populations based on the minimum stimulation frequency at which they were recruited. For example, the line labeled 10 Hz illustrates the average recruitment latency across stimulation frequencies for the 3 motor units recruited during 10 Hz stimulation. The effect of increasing stimulation frequency on decreasing recruitment latency is evident in each of these populations.

**FIGURE 3 | Number of units recruited and latency of discharge onset were dependent on stimulation frequency. (A)** The time between the beginning of stimulation and the onset of motor unit firing is illustrated for a single motor unit at three stimulation frequencies. The solid horizontal line shows the duration of the stimulation. **(B)** Higher stimulation frequencies recruited more motor units, with significant differences (∗p < 0.05) between the indicated stimulation frequencies. **(C)** The change in recruitment latency

#### **TEMPORAL RELATIONSHIP BETWEEN STIMULATION PULSES AND SUBSEQUENT DISCHARGES**

Once motor units were recruited, their discharge rate increased until a steady-state was reached, as shown for a single motor unit in **Figure 4A**. Motor unit discharge rate was not proportional to stimulation frequency, as the discharge rate reached approximately the same peak level regardless of stimulation frequency. Peak motor unit discharge rates were compared for the 9 motor units that were recruited during 20, 30 and 40 Hz stimulation. Peak discharge rate was significantly lower, albeit only slightly, with a stimulation frequency of 20 Hz (motor unit discharge rate = 7.8 ± 1.1 Hz) than with stimulation frequencies of 30 Hz (8.6 ± 1.1 Hz) or 40 Hz (8.4 ± 1.1 Hz), which were not significantly different (**Figure 4B**).

Qualitatively, motor units appeared to fire asynchronously from the stimulation pulses, as the latency between stimulus pulses and motor unit action potentials was not constant (**Figure 5A**, see also **Figure 1B**). PSTH analysis revealed that

with stimulation frequency is illustrated for each of the 25 motor units. The minimal stimulation frequency required to recruit each motor unit varied. **(D)** The 25 motor units were grouped by the minimal stimulation frequency required for their recruitment, and the average recruitment latencies were calculated for each group. Higher stimulation frequencies recruited motor units at shorter latencies, with significant differences (∗p < 0.05) between the indicated stimulation frequencies.

motor units fired at an H-reflex latency (1–2 ms peak ∼35–44 ms after a stimulation pulse) more often than would be expected from a completely asynchronous distribution, as illustrated in **Figure 5B** for a single motor unit. This analysis was performed for all trials in which a motor unit was recruited by a stimulation frequency of 10 or 20 Hz. In these 12 trials (*n* = 9 motor units), 24% (range 18–58%) of the motor unit discharges occurred at an H-reflex latency after a stimulation pulse, compared to the ∼2– 4% of the time that would indicate true asynchronous firing.

The probability of a stimulation pulse causing a motor unit to fire at an H-reflex latency was dependent upon the timing of the pulse. The "after firing period", the time between the stimulation pulse and the previous motor unit discharge, was calculated for all motor units recruited by 10 or 20 Hz stimulation (12 trials, 9 motor units), as illustrated for a single motor unit in **Figure 6A**. Averaged across motor units, stimulation pulses delivered within 50 ms after a motor unit discharge (after firing period <50 ms) never caused the motor unit to fire at an H-reflex

**FIGURE 4 | Motor unit discharge rate was not proportional to stimulation frequency. (A)** As illustrated for a single motor unit activated by three stimulation frequencies, there is a gradual increase in discharge rate, which then reaches a steady-state. The solid horizontal line shows the duration of the stimulation. **(B)** Averaged across subjects, the peak discharge rate was significantly (∗p < 0.05) lower during 20 Hz stimulation than during 30 or 40 Hz stimulation. **(C)** Averaged across subjects and stimulation frequency, motor unit discharge rate during the first second of sustained firing was significantly (∗p < 0.05) lower than the discharge rate during the last second of stimulation.

latency (**Figure 6B**). As the after firing period grew longer, the probability of the motor unit firing at an H-reflex latency after a stimulation pulse increased up to a maximum of 0.63 with an after firing period of 92 ms. Again, this analysis was performed for the 12 trials in which a motor unit was recruited by a stimulation frequency of 10 or 20 Hz.

#### **INSTANTANEOUS DISCHARGE RATE AFTER CESSATION OF ELECTRICAL STIMULATION**

Motor units often continued to fire after stimulation had ended, with sustained firing for at least one second occurring in 102 of 115 trials (89%). For the 9 motor units recruited during 20, 30 and 40 Hz stimulation, the discharge rate during the last second of stimulation (7.9 ± 1.1 Hz) was significantly higher than the discharge rate during the first second of sustained firing (5.8 ± 1.8 Hz) after stimulation ended (**Figure 4C**). The duration of the sustained activity was not quantified, as subjects were instructed to relax completely in order to allow 2 min of rest between trials and limit experiment duration. Although subjects stated that they were relaxed and were not voluntarily contracting, instructing them to "relax completely" terminated the motor unit activity.

#### **DISCHARGE AT THE TIME OF RECRUITMENT OF ADDITIONAL MOTOR UNITS**

In 10 trials, two clearly distinguishable motor units were recruited during the stimulation, as illustrated in **Figure 7A**. The discharge rate of the first motor unit recruited ("control unit") followed the pattern described above, with an initial increase followed by a steady-state in discharge rate. This discharge rate then remained constant even as additional motor units ("test units") were subsequently recruited. In no trials did the discharge rate of a control unit increase as a test unit was recruited. Across the ten

analyzed paired motor unit recordings, the control unit discharge rate during the 1 s period immediately prior to the recruitment of the test unit was not significantly different than the control unit discharge rate during the subsequent 1 s period (ANOVA, *p* > 0.05; **Figure 7B**). The discharge rates of both the control and test units decreased significantly once the stimulation ended (ANOVA, *p* < 0.05; **Figure 7C**).

#### **DISCUSSION**

Repetitive electrical stimulation of the tibial nerve, delivered at an intensity that was below threshold for producing H-reflexes (or M-waves) with a single pulse, resulted in the gradual recruitment of low threshold soleus motor units. A wide range of relatively high electrical stimulation frequencies resulted in a narrow range of relatively low motor unit discharge frequencies. The electrically-evoked afferent volley influenced motoneuron discharge "directly", resulting in motor unit discharge that was time-locked to the stimulus pulses as Hreflexes, and "indirectly", as reflected by motor unit discharge that was temporally uncoupled from the stimulation. Accordingly, the discharge of recruited motor units was consistent with the physiological activity that occurs during voluntary contractions, including recruitment according to Henneman's size principle, relatively low discharge rates and asynchronous firing.

#### **ELECTRICAL STIMULATION RECRUITED LOW THRESHOLD MOTOR UNITS**