human stretch reflex pathways reexamined - university of

21-3-2019 Human stretch reflex pathways reexamined

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3921411/?report=printable 1/24

J Neurophysiol. 2014 Feb 1; 111(3): 602–612.Published online 2013 Nov 13.doi: 10.1152/jn.00295.2013: 10.1152/jn.00295.2013

PMCID: PMC3921411PMID: 24225537

Human stretch reflex pathways reexaminedŞ. Utku Yavuz, Natalie Mrachacz-Kersting, Oğuz Sebik, M. Berna Ünver, Dario Farina, and Kemal S. Türker

Department of Neurorehabilitation Engineering, Bernstein Focus Neurotechnology Göttingen, Bernstein Center forComputational Neuroscience, University Medical Center Göttingen, Georg-August University, Göttingen, Germany;Center for Sensory-Motor Interaction, Department of Health Science and Technology, Aalborg University, Aalborg,

Denmark;Koç University School of Medicine, Sariyer, Istanbul, Turkey; andEge University Faculty of Medicine, Biophysics Department, Izmir, TurkeyCorresponding author.

Address for reprint requests and other correspondence: K. S. Türker, Koç Univ. School of Medicine, RumelifeneriYolu, 34450 Sariyer, Istanbul, Turkey (e-mail: [email protected]).

Received 2013 Apr 24; Accepted 2013 Nov 8.

Copyright © 2014 the American Physiological Society

AbstractReflex responses of tibialis anterior motor units to stretch stimuli were investigated in human subjects.Three types of stretch stimuli were applied (tap-like, ramp-and-hold, and half-sine stretch). Stimulus-induced responses in single motor units were analyzed using the classical technique, which involvedbuilding average surface electromyogram (SEMG) and peristimulus time histograms (PSTH) from thedischarge times of motor units and peristimulus frequencygrams (PSF) from the instantaneous dischargerates of single motor units. With the use of SEMG and PSTH, the tap-like stretch stimulus induced fiveseparate reflex responses, on average. With the same single motor unit data, the PSF technique indicatedthat the tap stimulus induced only three reflex responses. Similar to the finding using the tap-like stretchstimuli, ramp-and-hold stimuli induced several peaks and troughs in the SEMG and PSTH. The PSFanalyses displayed genuine increases in discharge rates underlying the peaks but not underlying thetroughs. Half-sine stretch stimuli induced a long-lasting excitation followed by a long-lasting silent periodin SEMG and PSTH. The increase in the discharge rate, however, lasted for the entire duration of thestimulus and continued during the silent period. The results are discussed in the light of the fact that thedischarge rate of a motoneuron has a strong positive linear association with the effective synaptic current itreceives and hence represents changes in the membrane potential more directly and accurately than theother indirect measures. This study suggests that the neuronal pathway of the human stretch reflex does notinclude inhibitory pathways.

Keywords: human, EMG, muscle spindle reflex, peristimulus time histogram, peristimulus frequencygram

�� in 1924 observed that a lengthening of a muscle will cause that muscle tocontract (Liddell and Sherrington 1924). This behavior is also referred to as the stretch reflex and has sincebeen utilized as both a diagnostic and research tool (Marsden et al. 1976; Matthews 1986). In the former,the integrity of the nervous system along the various levels of the spinal cord may be tested when a spinalcord lesion is suspected. As a research tool, the reflex has been evoked to gain an understanding of theunderlying neural pathways involved (Dietz and Sinkjaer 2007).

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There are several techniques to impose a lengthening of the target muscle. The most recent studies usingjoint rotation perturbations have applied ramp-and-hold-type stimuli; many other types of stimuli,including tapping the tendon using hand-held devices, feedback control tappers, and sine waves have alsobeen implemented (Beith and Harrison 2004; Burke and Schiller 1976; Frigon et al. 2011; Mrachacz-Kersting et al. 2006; O'Sullivan et al. 1998; Suresh et al. 2005; van Doornik et al. 2009). The responserecorded in the target muscle is dependent on the technique used. For example, following a tap to theAchilles tendon, a single synchronous peak occurs (Gandevia et al. 1986), whereas ankle joint rotation willresult in several synchronous peaks (Sinkjaer et al. 1988). Other than the peaks in the EMG, there are alsosilent periods between the peaks that can only be observed if the studied muscle has some backgroundactivity preceding the stretch stimulus (Matthews 1984; Miles et al. 1995; Poliakov and Miles 1994).

To date, the methods used to study stretch reflexes are the probabilistic methods based on trigger-averagedsurface electromyography (SEMG) and the peristimulus time histogram (PSTH). Fundamentally, trigger-averaged SEMG and PSTH present the correlation of gross muscle and single motor unit activity againstthe stimulus timing, respectively. Whereas the SEMG is a conventional method that is also used forclinical diagnosis, the lesser known PSTH method is a histogram that shows the number of motor unitspike occurrences in a particular time window around the stimulus (Davey et al. 1986). However, recently,there have been a number of studies suggesting that the probabilistic methods distort the data significantlyand mislead the investigator regarding the sign, amplitude, and shape of the stimulus-evoked net synapticpotential underlying the reflex response (Awiszus et al. 1991; Türker and Cheng 1994; Türker et al. 1996).To test these assertions directly, known currents have been injected into regularly discharging motoneuronsin rat brain slices and the spike output analyzed using the probabilistic methods (Türker and Powers 1999,2003). These experiments illustrated that the probability-based methods, such as PSTH and SEMG, can bemisleading in representing the input (synaptic) current. The underlying reason is that these methods areinfluenced not only by the input current but also by the autocorrelation factor of the underlying spikes(Türker and Powers 1999, 2003, 2005). The action potentials, which are advanced or delayed withexcitatory or inhibitory postsynaptic potentials (PSPs), occur again synchronously after one interspikeinterval. Since these probabilistic methods rely on the probability of spike occurrence, these consecutiveresponses can be erroneously interpreted as long- or medium-latency reflex responses.

In the brain slice experiments, another method was also tested that has been claimed to overcome theautocorrelation/synchronization-related errors: peristimulus frequencygram (PSF; Türker and Cheng1994). The basic theory that underlies the PSF method stands on the assumption that there is a positivelinear relationship between input current into a motoneuron and its discharge rate (Powers et al. 1992). ThePSF method simply plots the instantaneous discharge rates of single motor units against the time of thestimulus. The brain slice experiments have demonstrated that the time course of the net input current to amotoneuron is more accurately illustrated using the PSF method compared with the probabilistic methods(Türker and Powers 1999, 2003, 2005).

The aim of this work was to use both the probabilistic (SEMG and PSTH) and discharge rate (PSF)-basedmethods to reinvestigate the time course of the postsynaptic potential in human tibialis anterior (TA)motoneurons induced by various stretch stimuli. We expect that some of the reflex responses “established”using probabilistic methods might be synchronization-related artifacts. We have also examined differentstimulation methods of inducing stretch reflexes to test that our comparison of analysis methods would beapplicable to various forms of inducing stretch reflexes in human subjects and also to test the claim thatthe different forms of inducing stretch reflex may initiate varying number of reflex responses (Gandevia etal. 1986; Sinkjaer et al. 1988). First, we hypothesize that the shape and the speed of the stimulus willdetermine the properties of the reflex responses. Second, we propose that the PSF analysis will identifydifferent numbers of reflex responses compared with the probabilistic analyses.

METHODSThe experiments were approved by the Human Ethics Committee of the University of Aalborg (N-20090019), and the participants gave written informed consent according to the Declaration of Helsinki.The reflex responses of 107 low-threshold (<20% maximal voluntary contraction; 4–19% as a range ofmaximum EMG and 12–38% as a range of maximum force) TA motor units were recorded in 12volunteers (age 31 ± 5 yr; range 25–40 yr). The SEMG and potentials from single TA motor units (SMU)



Surface EMG.

Single motor units.

Surface EMG.

Single motor units.

and the force from the footplate were recorded on a computer using the CED interface (CambridgeElectronic Design, Cambridge, UK) for off-line analysis. The SMUs were recruited by isometriccontraction of the muscle as the foot was dorsiflexed against a foot pedal, which was attached to a forcetransducer. Subjects contracted their muscles to activate one or two motor units on the single-unitelectrode. They were provided with the feedback of the activity of the most prominent unit with the largestmotor unit action potential and asked to keep the discharge rate of the selected unit at a rate of ∼6 Hz usingthe sound of the accepted unit pulses. While the subject maintained the background level of excitation ofthe muscle's motoneuron pool at a constant level, stretch stimuli were applied and reflex responses ofsimultaneously recorded surface EMG and SMUs were recorded. Briefly, the procedures outlined belowwere used.

Recordings

Surface EMG of the right TA was recorded using bipolar electrodes placed 2 cm apartaccording to the recommendations of Garland et al. (1994). The skin overlying the muscle was cleanedusing 70% alcohol to reduce electrode resistance to below 5 kΩ. The signals were amplified 1,000 times,bandpass filtered with a cutoff frequency of 20–500 Hz, and recorded on the CED system. Data wereanalyzed using the software available in the CED Spike2 data acquisition system.

To record motor unit activity, three pairs of Teflon-insulated silver wires (75 μm incore diameter) were inserted into the muscle with 25-gauge needles; the needles were then withdrawn,leaving the wires in the belly of the muscle. In most experiments, stable recordings of one or two unitswere obtained from each of the insertion sites. The shape of the SMU action potential was discriminatedonline using a template-matching algorithm (version 6; Spike2), which generated a recognition pulsewhenever it matched the shape of an action potential. Single-unit potentials were sampled at 20,000 Hzand recorded on a computer for off-line analyses (data acquisition and analysis system, CED Spike2).

Stretch Stimulus

The right leg was fastened to a servo-controlled hydraulic actuator (model 215.35; MTS Systems) (Voigt etal. 1999). The angle of the ankle was arranged to the anatomical rest position of the subject, and thesubject chair was arranged to extend the knee angle to 120°. The foot segment of the right leg of thesubject was firmly strapped to a custom-made plate that extended from the actuator, thus producing a tightinterface between the arm of the motor and the foot of the subject, ensuring that the movement of theactuator was transmitted solely to the ankle joint (Fig. 1). Also, the subject's thigh was strapped to the chairto ensure that movement of the actuator did not change the knee and hip angle. The angular position of theactuator was monitored by an angular displacement transducer and feedback controlled using aproportional integral derivative (PID) control system (DC ADT series 600; Transtek). Stretches wererandomly applied at intervals ranging between 5 and 7 s. Three different types of perturbations wereimposed randomly for each subject: a tap, a ramp-and-hold, and a half-sine perturbation (Fig. 2). Theangular velocity, amplitude, and duration were 100°/s, 1°, and 0 ms for tap perturbations and 200°/s, 4°,and 460 ms for ramp-and-hold perturbations; amplitude and duration were 1° and 100 ms for half-sineperturbations.

Analyses

Raw SEMG records were full-wave rectified and averaged 500 ms before and 250 ms afterthe imposed perturbation, spanning an analysis time of 750 ms. Cumulative sum (CUSUM; Ellaway 1978)was calculated from the averaged SEMG to illustrate the exact timing and the size of the reflex response.Any poststimulus deflection that is larger than the maximal prestimulus deflections (error box) and appearsbefore the reaction time to the perturbations was considered a genuine (significant) reflex response to thestimulus (Brinkworth and Türker 2003; Türker et al. 1997).

Single motor units were identified using the Spike2 program (CED), and each unitwas separated using the program's template-matching algorithm. PSTHs and PSFs were then constructedfrom the times of occurrence of action potentials of individual motor units. To build the PSTH, firing timesof each selected unit were converted into acceptance pulses by the Spike2 program and placed into 1-msbins around the time of the perturbation stimulus. Once acceptance pulses belonging to an SMU are placed

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Statistical analyses.

in these bins during 100 or more triggers, the end product, the PSTH of that unit, indicates the number ofoccurrences of spikes at each of the bins around the time of the stimulus. PSF is the superimposition of theinstantaneous discharge rates of a selected unit around the time of the stimulus and indicates theexcitability of the motoneuron membrane (Powers and Türker 2010; Türker and Powers 2005). To buildthe PSF, acceptance pulses from Spike2 program were turned into instantaneous discharge rate values.Instantaneous discharge rate values obtained using 100 or more triggers were superimposed around thetime of the stimulus.

CUSUMs for the PSTH and PSF graphs were also obtained to make any subtle but persistent changesdetectable. CUSUM of the PSF highlights the changes in the discharge rate after the stimulus by addingthe differences in the bin values from the average prestimulus bin value (Türker et al. 1996). This isessentially the same approach as the original CUSUM that was built for the PSTH (Ellaway 1978). Theonly difference is that the CUSUM of the PSTH uses the number of counts in each bin, whereas theCUSUM of the PSF uses average discharge rate values in each bin.

The reflex responses of one or two units were determined from each electrode off-line. The chosen unitswere always the ones with the largest action potentials because they were easier to identify and followonline during the experiment. The motor unit potentials were high-pass filtered at around 1,000 Hz tofurther clarify the shape and amplitude of the unit of interest and to depress the amplitude of all other unitsincluding low-frequency noise in the record. This process reduces the chances of superimposition of otherunit potentials onto the action potential of the unit of interest. Such superimpositions can interfere with therecognition of the unit potential by the template-matching program and hence undermine the estimation ofthe reflex response. Other than using filtering techniques to highlight the unit of interest clearly, we havealso used two other approaches in this experiment so that recognition of the unit throughout the experimentwould be possible. These were the limitation imposed on the stimulus intensity and the number of units ina given intramuscular electrode. In an ideal setting, the electrode contained only one motor unit with alarge signal-to-noise ratio and the stimulus intensity did not recruit other (previously nonactive) units at thereflex latency. If these two conditions were met, reliable and repeatable results were obtained.

Following these somewhat limiting preconditions, the amplitude of the imposed perturbation was fixed to1° for the tap stimulus, 4° for the ramp-and-hold stimulus, and 1° for the half-sine stimulus. Theseamplitudes were used because they recruited at least 2 spikes at the onset of the short-latency response tothe imposed stretch in 10 consecutive stimuli. Usually, our aim was 2–4 spikes at the reflex latency (out of10 stimuli), equivalent to an excitatory postsynaptic potential (EPSP) size of about 2–4 mV (Ashby andZilm 1978; Miles et al. 1989), while setting the perturbation amplitude in an experiment. Any furtherincrease in the perturbation amplitude often recruited extra motor units at the stretch reflex latency andhence interfered with the recognition of the selected motor unit at this latency. High contraction levels alsocould not be studied because they often recruit many more motor units, which are also reflected in theintramuscular electrode recordings. When an intramuscular EMG record has many units, the stimulusintensity that can induce stretch reflexes in SEMG would induce many superimpositions of motor unitpotentials at the reflex latency and hence would not allow for an accurate study of the PSPs to singlemotoneurons.

At this level of intensity, several hundred stimuli were delivered to quantify the net response to thestimulus. To achieve this objective in this study, the stimuli continued to be delivered as long as the shapeof the motor unit potentials was distinctly recognized and for as long as the subject was able to fire the unitregularly. The units that were lost before delivering at least 100 stimuli were not analyzed further. Morethan 100 stimuli (279 ± 166; range 100–986) were delivered for each of the 107 units reported in thisarticle. Since the number of stimuli varied between the units, we normalized the size of the reflexresponses to the number of triggers used. Hence we obtained values such as extra spikes per trigger orextra Hz per trigger (see Tables 1–3). Latencies were determined from the turning point of any of thesignificant deflections in the CUSUM records. Amplitudes of the reflex responses were determined usingthe method described by Brinkworth and Türker (2003). In essence, the reflex response was normalized tothe maximum possible inhibition (no active units for the entire duration of the reflex) and marked as thepercentage of this maximum inhibition.

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The latencies of responses between analysis techniques were investigated using one-way ANOVA. Thedifferences between each group were tested using Fisher's least significant difference post hoc test. Also,the amplitude of responses between tap-like and ramp-and-hold stimulation types was investigated usingone-way ANOVA. The incidence rates of responses were compared between SEMG, PSTH, and PSFmethods for each stimulation method (tap, ramp and hold) using the McNemar statistical test. Theprevalence values of M1, M2, and M3 for different SMUs were grouped according to their prestimulusaverage discharge rates into three groups as low (6–8 Hz), middle (8–10 Hz), and high (10–12 Hz)discharge rate. The differences between these groups were compared using the χ statistical test for thePSTH and PSF analysis methods. For all statistical analyses, the null hypothesis was rejected if probabilitywas <0.05.

RESULTSReflex responses to a minimum of 100 and a maximum of 986 imposed perturbations of 107 units wereanalyzed. Three types of stretch stimuli were applied, and results were analyzed using three differentmethods. The stimuli were simple tap-like stretch, ramp-and-hold stretch, and half-sine stretch. Stimulus-induced responses in SEMG and SMUs were analyzed using the classical probability-based technique,which involved building rectified averaged SEMG around the time of the stimulus and constructingPSTHs from the firing times of the single units. The same SMU data were also analyzed using PSFs fromthe instantaneous discharge rates of the units.

Simple Tap-Like Stretch Stimulus/Perturbation

Tap-like stimulus was applied to 12 subjects, and 72 motor units were recorded. Using PSTH as theindicator, we found that the tap-like stretch stimulus induced five separate significant responses: the firstresponse was a short-latency excitatory response (M1), or the short-latency stretch reflex; the second was asilent period (SP1); the third was a medium-latency excitation, or M2; the fourth was a long-latency silentperiod (SP2); and the fifth was a broad peak (M3) (Table 1 and Fig. 3). With the use of the same motorunit data, the PSF technique indicated that the tap-like perturbation induced only three significant reflexresponses corresponding with the M1, M2, and M3 responses on the SEMG and PSTH (Table 1). The PSFdisplayed significant increases in the discharge rate of the unit during the first excitatory reflex in thePSTH. Although the second (M2) and the third (M3) responses were rarely observed in the same record(10 in PSF and 18 in PSTH over 72 motor units), when they did occur the discharge rate of the unitunderlying these responses increased significantly. On the other hand, discharge rate did not decreasesignificantly during either SP1 or SP2 (Table 1), hence suggesting that these silent periods do not indicateinhibitions.

Ramp-and-Hold Stretch Stimulus

In 16 units in 11 subjects, ramp-and-hold stretch stimuli/perturbations were applied. In all experiments,this type of stimulus induced several peaks and troughs in the SEMG and PSTH similar to the responses tothe tap-like stretch stimuli. PSF illustrated genuine increases in the discharge rates underlying most of thepeaks in the SEMG and PSTH (Fig. 4). Again, there was no significant decrease in discharge rateunderlying the silent periods that were observed in SEMG and PSTH records. Latencies and amplitudes ofthe reflex responses are given in Table 2.

Half-Sine Stretch Stimulus

Half-sine stimulus was applied to 8 subjects, and 19 motor units were recorded from those subjects. Thesestimuli induced a long-lasting excitation (LLE) followed by a long-lasting silent period (LLSP) in SEMGand PSTH. The increase in the discharge rate (as displayed in the PSF records), however, lasted for theentire duration of the mechanical stimulus and continued even during the phase where the number ofspikes decreased in the SEMG and PSTH (Fig. 5). Table 3 details the latencies and amplitudes of the reflexresponses elicited using half-sine stretch stimuli.

Comparison of Latency, Duration, and Amplitude of Reflex Responses

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No differences were found between the latencies of the M1 responses determined using the two differentanalysis methods. However, the response amplitudes that were induced by ramp-and-hold stimuli werealways significantly stronger than the ones induced by tap-like stimuli for all analysis techniques. Whendurations of responses were compared for the tap-like stimulation, it was found that the durations of M2and M3 responses were longer when determined using the PSF technique compared with the durationsdetermined using the SEMG and PSTH analyses. However, for the ramp-and-hold stimulation type, theonly significant difference was found in M2 responses between the durations calculated by PSF comparedwith the other techniques (SEMG, PSTH; Table 2). The latency of LLE in half-sine stimulation wascompared with the latency of M1 responses that were induced with the tap-like and the ramp-and-holdstimulations. Results demonstrated that the latency of LLE was significantly longer than the latency of M1responses for SEMG, PSTH, and PSF analyses.

Comparison of the Number of Reflex Responses

The responses to the tap-like stretch and the ramp-and-hold stretch stimuli often generated similar numbersof reflex responses. PSF analyses indicated that only the M1, M2, and M3 responses on the SEMG andPSTH were genuine excitatory events, since the discharge rate of single units significantly increasedduring these periods. The differences between incidence rates of excitatory responses in each of theanalysis methods were quantified using the McNemar test. The first excitatory response was observed inalmost every motor unit for each method (for tap-like stretch: SEMG 93%, PSTH 88%, PSF 83%; forramp-and-hold stretch: SEMG 93%, PSTH 88%, PSF 88%). For the tap-like stimulus, the incidence rate ofM2 in PSF (67%) was significantly higher than that in SEMG (32%) and PSTH (37%) (P < 0.05 for bothpairs). In contrast with M2, the incidence rate of M3 was significantly higher in SEMG (80%) and PSTH(68%) compared with that in PSF (30%) (P < 0.05 for both comparisons; Table 1). Thus the incidence ofreflex responses was one of the differences between the probability- and discharge rate-based analysismethods. The other major difference between the methods was the incidence of the silent period responses.The tap-like and ramp-and-hold stretch stimuli induced silent periods that followed the M1 and M2responses in the classical analysis methods, whereas no such periods were observed in the PSF technique (Fig. 6).

The prevalence of the M1 and M2 responses depended on the discharge rate of the units. As thebackground discharge rate increased, the prevalence of the M2 responses also increased, whereas theincidence of the M1 response decreased, in the PSTH records (Fig. 7). The PSF records also indicated anincrease in the prevalence of the M2 response with the increase in the background discharge rate of theunit. The amplitudes of these responses were also related to the background discharge rate of theunderlying units.

The significance of the difference between the three discharge groups (low, medium, and high) for theprevalence values for M1, M2, and M3 was compared using the χ test. The null hypothesis was that theprevalence does not depend on the discharge rate of units (χ = 5.990 for 2 degrees of freedom). For thePSTH method, no significant differences were found for the prevalence values of M1 and M3 for any ofthe three different discharge rates (M1: P > 0.05, χ = 0.293; M3: P > 0.05, χ = 1.659). However, asignificant difference was found for the prevalence values of M2. For the PSF method, no significantdifferences were found for the prevalence values of M1 among groups (P > 0.05, χ = 0.869). On theother hand, the prevalence values for M2 and M3 were significantly different among at least one pair ofdischarge rate groups (M2: P < 0.05, χ = 5.996; M3: P < 0.05, χ = 7.539).

In one subject, the tap-like stretch stimulus induced only M2 in one unit, whereas another simultaneouslyrecorded unit displayed both M1 and M2 responses. This was a rare event and was only observed in 1 of72 units tested using this type of stretch stimulus.

DISCUSSIONThere are two main new findings in this study. First, rejecting our first hypothesis, this study has shownthat the tap-like stretch induced a similar number of reflex responses as the ramp-and-hold type stretchstimulus. Second, supporting our second hypothesis, whereas probabilistic analyses indicated several

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peaks and troughs in the records, discharge rate-based methods only confirmed the sign of excitatoryresponses.

Analysis Methods

In this study, we have used three methods to determine the reflex responses: rectified and averaged SEMG,PSTH, and PSF. The SEMG and PSTH methods are both conventional methods that have been used inreflex studies for ∼35 years. Both methods illustrate the density of motor unit spikes around the time ofstimulation (Ellaway 1978). With these methods, poststimulus periods with spike densities lower thanthose during prestimulus periods are labeled as inhibition, and those with higher densities are labeled asexcitation (Brooke et al. 1999; Okdeh et al. 1999; Sonnenborg et al. 2000). However, use of only theseclassical methods gives an incomplete picture of the synaptic connection for the following reasons. First,they rely on the number of motor unit spikes occurring at a fixed time from the stimulus. This number maybe misleading if part of the reflex response falls in the shadow of an earlier and more powerful reflexresponse (Awiszus et al. 1991; Türker and Cheng 1994). Second, a strong excitatory or inhibitory reflexresponse can synchronize the timing of the action potentials in relation to the stimulus. This synchronousdischarge can induce several peaks and troughs in both SEMG and PSTH as the motoneuron discharges ata regular rate after the initial reflex response (Awiszus et al. 1991; Miles and Türker 1987; Türker andCheng 1994). However, the PSF represents the instantaneous discharge rate of the motoneurons (Türkerand Cheng 1994). Because the discharge rate of a motoneuron directly represents the effective net currentinjected into it either slowly (Granit et al. 1963; Powers et al. 1992) or rapidly (Baldissera et al. 1982), anysignificant change in the discharge rate of a motoneuron as a result of a stimulus can be used to estimatethe stimulus-induced net synaptic potential (Türker and Powers 1999, 2003, 2005).

The CUSUM of the average records is also necessary because it amplifies small but persistent changes(Ellaway 1978). Because CUSUM indicates the area of responses, it can also be used to estimate therelative strength of reflex responses (Binboğa and Türker 2012; Brinkworth and Türker 2003).

Reflex Responses to Different Shape of Stimulation

The response recorded in the target muscle is dependent on the technique used (Gandevia et al. 1986;Sinkjaer et al. 1988). Moreover, it is known that depending on the shape, slope, and amplitude ofmechanical stimulation, different sensory afferents can be activated (Matthews 1984). An additionalcomplication arises when the same perturbation is applied to either a hand muscle or a lower limb muscle.Typically, in both instances various peaks may be observed, which have been labeled M1 and M2, with anadditional peak often seen in lower limb muscles (M3) (Petersen et al. 1998). Therefore, in the presentstudy, three different stimulation shapes (tap, ramp and hold, sinusoidal) were used to investigate thestretch reflex on TA muscle.

The only difference between these methods was the occurrence of silent periods that followed the M1, M2,and the LLE responses in the classical methods. These silent periods with a low number of actionpotentials were not recognized as periods of inhibitions in the PSF technique, since during these periodsthe discharge rates of action potentials were the same as during the prestimulus periods. Therefore, itwould be wrong to label these periods of low motor unit activity as inhibitory reflex responses. For aperiod to be labeled as an inhibitory period with the PSF method, the discharge rate of the underlyingmotor units needs to decrease below the prestimulus discharge rate values (Türker and Powers 1999, 2003,2005). We suggest that the low number of spikes was possibly caused by the phase advance of spikes as aresult of the stretch stimulus. The phase advancement generated the M1 response. The same phaseadvancement must also have applied to the SP2 period that induced the M2 response. Genuineness of theM2 response is clear because not only was it present in most units but also it became more prominent withthe increase in the discharge rate. In the lower discharge rates, it must be underestimated due to theshadowing effect of the M1 response.

For the TA muscle it is known that the M1 component arises from the activation of group Ia muscleafferents, which synapse directly onto the motoneuron (Darton et al. 1985; Grey et al. 2001). Theneurological basis of the M2 and M3 response, however, is not so clear and has been the subject of muchcontroversy (Burke et al. 1983; Christensen et al. 2001; Lee and Tatton 1978; Sinkjaer et al. 1999). Theorigin of M2 in hand muscles has been claimed to be cortical (Matthews et al. 1990), whereas the M2



response in lower limb muscles occurs too fast to be cortical (Darton et al. 1985; Thilmann et al. 1991).Reports have suggested a group Ib or group II pathway may be responsible for the M2 response (Dietz1998; Dietz et al. 1985; Grey et al. 2001; Schieppati and Nardone 1997). The origin of the M3 responsehas been claimed to be via a group Ia pathway, which traverses the motor cortex (Petersen et al. 1998). It isalso possible that the M2 and M3 responses may be due to the synchronous reoccurrence of spikes afterthe M1 response (Türker and Powers 2003). Although several studies have deliberated possible pathwaysfor the excitatory responses, only a few studies have reported silent period in the SEMG or PSTH after theM1 response (Matthews 1984; Miles et al. 1995; Poliakov and Miles 1994). Moreover, silent period afterthe M2 response has never been reported. The reasons for not recognizing the silent periods in earlier workmay be twofold. First, most previous stretch reflex studies involved relaxed muscles, which makeobserving inhibitory responses difficult (Avela et al. 1999; Fellows et al. 1993; Thilmann et al. 1991;Yamamoto et al. 2000). In the present study, the subjects maintained a fixed level of excitation of theirmuscles to make sure that the excitatory as well as the inhibitory pathways could be identified if theyexisted. The other possible reason for not recognizing the silent periods, even when the existence of suchperiods was possible when a low spike density was used as the criterion (as in SEMG and PSTH methods),may have been the fact that most did not use analysis methods that brought out subtle but consistentchanges in the SEMG or PSTH, such as the CUSUM technique (Darton et al. 1985; Edin and Vallbo 1990;Grey et al. 2001; Mrachacz-Kersting et al. 2006; Petersen et al. 1998; Schuurmans et al. 2009; Thilmann etal. 1991).

In the present study, we also used the sinusoidal stimulation to find evidence of the contribution of group IIafferents. It is known that group I and II afferents exhibit different dynamics (Cooper 1961; Matthew 1984;Matthews and Stein 1969). Whereas group II afferents are sensitive to changes in the length of muscle,group I afferents are activated by the acceleration in the force (Edin and Vallbo 1990; Matthews 1984). Inthis study the slow stretch (sinusoidal) stimulus induced two significant reflex responses in SEMG andPSTH, excitation followed by silent period. However, the PSF analysis has again differed from theprobabilistic analyses and indicated only one significant reflex, excitation. The latency of this excitationwas longer than the latency of the M1 response that was induced by ramp-and-hold and tap-likestimulations. These findings suggest that slow stretch-induced excitatory response may originate mainlyfrom the slower conducting group II afferents.

Effect of Discharge Rate on Prevalence of Reflexes

Prevalence of the M1 and M2 responses depended on the discharge rate of the units underlying theseresponses. As the background discharge rate increased, the prevalence of the M2 response increasedwhereas M1 decreased in the PSTH and PSF records. The size of these responses was also related to thebackground discharge rate of the underlying units. Whereas the M1 response amplitude decreased with theincrease in the background rate of the unit, the amplitude of the M2 response increased along with thedischarge rate. This is an evidence that the slope of the membrane potential trajectory becomes steeper asthe discharge rate increases (Ashby and Zilm 1982; Miles and Türker 1987; Miles et al. 1989), henceallowing the appearance of the long-latency PSPs that were hidden behind earlier PSPs during the lowdischarge rates. Because the background discharge rate affected the prevalence of these responses, it is putforward that two responses are definitely genuine and that the M2 response is generally larger than the M1response but often appears smaller since it falls in the shadow of the earlier M1 response at lowcontraction levels (SEMG) or low discharge rates (PSTH and PSF).

An Odd Finding

In one subject, the tap-like stretch stimulus induced only M2 in one unit, whereas another simultaneouslyrecorded unit responded with both M1 and M2, which were also indicated in the SEMG. This was a rareevent and was only observed in 1 of 107 units tested. However, this may indicate that some units are onlyconnected with the spindle primary afferents via a long and/or polysynaptic pathway.

Limitations of Study



This study assumed that the increase in the discharge rate in PSF records indicates genuine excitatoryconnections. Recently, however, Piotrkiewicz and Kudina (2012) put forward that not all increases in thedischarge rates could indicate excitation. Using simulation studies and very short-lasting EPSPs, theyshowed that the pulse-like EPSPs prefer slower discharging phases of a unit and hence effectively separatethe higher discharge rate events from the lower discharge rate events. This generates an excitation-likegathering of high discharge rate events immediately following an excitatory event. However, we haveshown that this separation of discharges using pulse-like EPSPs does not occur in longer lasting EPSPs,since the discharge rates after the stimulus actually rise above the prestimulus rates (Türker and Powers1999). To be on the safe side, in this study we have considered a PSF event as significant only when thedischarge rate was over and above the highest prestimulus discharge rate and lasted long enough to allowits CUSUM to reach a level of significance (��).

The other limitation of the current experimental protocol was that we could not increase the stimulusintensity to generate large reflex responses because of superimposition problems when many units arerecruited at the reflex latency. High contraction levels also could not be studied because they oftenrecruited several motor units, which were also reflected in the intramuscular electrode recordings.

Concluding Remarks

The aim of this work was to use both the probabilistic (SEMG and PSTH) and discharge rate (PSF)-basedmethods to investigate the time course of the postsynaptic potential in human tibialis anterior motoneuronsinduced by the stretch stimuli. We showed earlier that the discharge rate of a motoneuron is a betterrepresentative of the postsynaptic potential than the classically used probabilistic methods. However, sincethe current knowledge on the stretch receptor pathways are “established” using only the probabilisticmethods, we wanted to analyze the same reflex data using both of these methods so that we could comparethe two methods and possibly revise some of the current beliefs. The present study indicates that theneuronal pathways of the stretch reflex may include up to three phases of excitations and excludeinhibitory pathways.

GRANTSThis study is supported by Marie Curie Chair Project (GenderReflex) MEX-CT-2006-040317, TurkishScientific and Technological Research Organization Grant TUBITAK-107S029-SBAG-3556, EuropeanResearch Council Advanced Grant DEMOVE 267888, and the Det Obelske Familiefond. S. U. Yavuz, O.Sebik, and M. B. Ünver are supported by Danish Government Scholarship Cultural Agreements 2010/11.K. S. Türker is a Fellow of the Turkish Academy of Sciences Association.

DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONSS.U.Y., N.M.-K., O.S., M.B.U., and K.S.T. performed experiments; S.U.Y., O.S., and M.B.U. analyzeddata; S.U.Y., N.M.-K., O.S., D.F., and K.S.T. interpreted results of experiments; S.U.Y., O.S., and M.B.U.prepared figures; S.U.Y., N.M.-K., O.S., D.F., and K.S.T. edited and revised manuscript; S.U.Y., N.M.-K.,O.S., M.B.U., D.F., and K.S.T. approved final version of manuscript; K.S.T. conception and design ofresearch; K.S.T. drafted manuscript.

REFERENCES

Ashby P, Zilm D. Characteristics of postsynaptic potentials produced in single human motoneurons byhomonymous group 1 volleys. Exp Brain Res 47: 41–48, 1982 [PubMed: 6288433]

Ashby P, Zilm D. Synaptic connections to individual tibialis anterior motoneurones in man. J NeurolNeurosurg Psychiatry 41: 684–689, 1978 [PMCID: PMC1083381] [PubMed: 681955]

Avela J, Kyröläinen H, Komi PV. Altered reflex sensitivity after repeated and prolonged passive musclestretching. J Appl Physiol 86: 1283–1291, 1999 [PubMed: 10194214]



Awiszus F, Feistner H, Schäfer SS. On a method to detect long-latency excitations and inhibitions of singlehand muscle motoneurons in man. Exp Brain Res 86: 440–446, 1991 [PubMed: 1756817]

Baldissera F, Campadelli P, Piccinelli L. Impulse coding of ramp currents intracellularly injected intopyramidal tract neurones. Exp Brain Res 48: 455–458, 1982 [PubMed: 6295796]

Beith ID, Harrison PJ. Stretch reflex in human abdominal muscles. Exp Brain Res 159: 206–213, 2004[PubMed: 15258713]

Binboğa E, Türker KS. Compound group I excitatory input is differentially distributed to human soleusmotoneuron. Clin Neurophysiol 213: 73–86, 2012 [PubMed: 22608971]

Brinkworth RS, Türker KS. A method for quantifying reflex responses from intra-muscular and surfaceelectromyogram. J Neurosci Methods 122: 179–193, 2003 [PubMed: 12573477]

Brooke JD, McIlroy WE, Staines WR, Angerilli PA, Peritore GF. Cutaneous reflexes of the human legduring passive movement. J Physiol 518: 619–628, 1999 [PMCID: PMC2269424] [PubMed:10381606]

Burke D, Gandevia SC, McKeon B. The afferent volleys responsible for spinal proprioceptive reflexes inman. J Physiol 339: 535–552, 1983 [PMCID: PMC1199177] [PubMed: 6887033]

Burke D, Schiller H. Discharge pattern of single motor units in the tonic vibration reflex of human tricepssurae. J Neurol Neurosurg Psychiatry 39: 729–741, 1976 [PMCID: PMC492438] [PubMed: 956859]

Christensen L, Andersen J, Sinkjaer T, Nielsen J. Transcranial magnetic stimulation and stretch reflex inthe tibialis anterior muscle during human walking. J Physiol 531: 545–557, 2001[PMCID: PMC2278473] [PubMed: 11230526]

Cooper S. The responses of the primary and secondary endings of muscle spindles with intact motorinnervation during applied stretch. Exp Physiol 46: 389–398, 1961 [PubMed: 13881160]

Darton K, Lippold OC, Shahani M, Shahani U. Long-latency spinal reflexes in humans. J Neurophysiol53: 1604–1618, 1985 [PubMed: 4009235]

Davey NJ, Ellaway PH, Stein RB. Statistical limits for detecting change in the cumulative sum derivativeof the peristimulus time histogram. J Neurosci Methods 17: 153–166, 1986 [PubMed: 3762224]

Dietz V, Quintern J, Berger W. Afferent control of human stance and gait: evidence for blocking of group Iafferents during gait. Exp Brain Res 61: 153–163, 1985 [PubMed: 4085594]

Dietz V, Sinkjaer T. Spastic movement disorder: impaired reflex function and altered muscle mechanics.Lancet Neurol 6: 725–733, 2007 [PubMed: 17638613]

Dietz V. Evidence for a load receptor contribution to the control of posture and locomotion. NeurosciBiobehav Rev 22: 495–499, 1998 [PubMed: 9595560]

Edin BB, Vallbo AB. Dynamic response of human muscle spindle afferents to stretch. J Neurophysiol 63:1297–1306, 1990 [PubMed: 2141632]

Ellaway PH. Cumulative sum technique and its application to the analysis of peristimulus time histograms.Electroencephalogr Clin Neurophysiol 45: 302–304, 1978 [PubMed: 78843]

Fellows SJ, Ross HF, Thilmann AF. The limitations of the tendon jerk as a marker of pathological stretchreflex activity in human spasticity. J Neurol Neurosurg Psychiatry 56: 531–537, 1993[PMCID: PMC1015014] [PubMed: 8505646]

Frigon A, Johnson MD, Heckman CJ. Altered activation patterns by triceps surae stretch reflex pathwaysin acute and chronic spinal cord injury. J Neurophysiol 106: 1669–1678, 2011[PMCID: PMC3191838] [PubMed: 21734111]

Gandevia SC, Miller S, Aniss AM, Burke D. Reflex influences on muscle spindle activity in relaxedhuman leg muscles. J Neurophysiol 56: 159–170, 1986 [PubMed: 2943878]

Garland SJ, Gerilovsky L, Enoka RM. Association between muscle architecture and quadriceps femoris H-reflex. Muscle Nerve 17: 581–592, 1994 [PubMed: 8196700]

Granit R, Kernell D, Shortess GK. Quantitative aspect of repetitive firing of mammalian to neurons,caused by injected current. J Physiol 168: 911–931, 1963 [PMCID: PMC1359475] [PubMed:14072865]

Grey MJ, Ladouceur M, Andersen JB, Nielsen JB, Sinkjaer T. Group II muscle afferents probablycontribute to the medium latency soleus stretch reflex during walking in humans. J Physiol 534: 925–933, 2001 [PMCID: PMC2278750] [PubMed: 11483721]

Lee RG, Tatton WG. Long loop reflexes in humans: clinical applications. In: Cerebral Motor Control inMan: Long Loop Mechanisms, edited by Desmedt JE, editor. Basel: Karger, 1978, vol. 4, p. 334–341



Liddell EGT, Sherrington C. Reflexes in response to stretch (myotatic reflexes). Proc R Soc Lond B BiolSci 96: 212–242, 1924

Marsden CD, Merton PA, Morton HB. Stretch reflex and servo action in a variety of human muscles. JPhysiol 259: 531–560, 1976 [PMCID: PMC1309044] [PubMed: 957257]

Matthews PB, Farmer SF, Ingram DA. On the localization of the stretch reflex of intrinsic hand muscles ina patient with mirror movements. J Physiol 428: 561–577, 1990 [PMCID: PMC1181663] [PubMed:2231424]

Matthews PB. Evidence from the use of vibration that the human long-latency stretch reflex depends uponspindle secondary afferents. J Physiol 348: 383–415, 1984 [PMCID: PMC1199408] [PubMed:6232375]

Matthews PB. Observations on the automatic compensation of reflex gain on varying the pre-existing levelof motor discharge in man. J Physiol 374: 73–90, 1986 [PMCID: PMC1182707] [PubMed: 3746703]

Matthews PB, Stein RB. The sensitivity of muscle spindle afferents to small sinusoidal changes of length.J Physiol 200: 723–743, 1969 [PMCID: PMC1350524] [PubMed: 4237132]

Miles TS, Poliakov AV, Nordstrom MA. Responses of human masseter motor units to stretch. J Physiol483: 251–264, 1995 [PMCID: PMC1157886] [PubMed: 7776236]

Miles TS, Türker KS, Le TH. Ia reflexes and EPSPs in human soleus motor neurones. Exp Brain Res 77:628–636, 1989 [PubMed: 2806452]

Miles TS, Türker KS. Decomposition of the human electromyogramme in an inhibitory reflex. Exp BrainRes 65: 337–342, 1987 [PubMed: 3556462]

Mrachacz-Kersting N, Grey MJ, Sinkjaer T. Evidence for a supraspinal contribution to the humanquadriceps long-latency stretch reflex. Exp Brain Res 168: 529–540, 2006 [PubMed: 16240144]

Okdeh AM, Lyons MF, Cadden SW. The study of jaw reflexes evoked by electrical stimulation of the lip:the importance of stimulus intensity and polarity. J Oral Rehabil 26: 479–487, 1999 [PubMed:10397180]

O'Sullivian MC, Miller S, Ramesh V, Conway E, Gilfillan K, McDonough S, Eyre JA. Abnormaldevelopment of biceps brachii phasic stretch reflex and persistence of short latency heteronymousreflexes from biceps to triceps brachii in spastic cerebral palsy. Brain 121: 2381–2395, 1998 [PubMed:9874488]

Petersen N, Christensen LO, Morita H, Sinkjaer T, Nielsen J. Evidence that a transcortical pathwaycontributes to stretch reflexes in the tibialis anterior muscle in man. J Physiol 512: 267–276, 1998[PMCID: PMC2231172] [PubMed: 9729635]

Piotrkiewicz M, Kudina L. Analysis of motoneuron responses to composite synaptic volleys (computersimulation study). Exp Brain Res 217: 209–221, 2012 [PMCID: PMC3282905] [PubMed: 22198533]

Poliakov AV, Miles TS. Stretch reflexes in human masseter. J Physiol 476: 323–331, 1994[PMCID: PMC1160444] [PubMed: 8046646]

Powers RK, Robinson FR, Konodi MA, Binder MD. Effective synaptic current can be estimated frommeasurements of neuronal discharge. J Neurophysiol 68: 964–968, 1992 [PubMed: 1432061]

Powers RK, Türker KS. Estimates of EPSP amplitude based on changes in motoneuron discharge rate andprobability. Exp Brain Res 206: 427–440, 2010 [PubMed: 20862458]

Schieppati M, Nardone A. Medium-latency stretch reflexes of foot and leg muscles analysed by coolingthe lower limb in standing humans. J Physiol 503: 691–698, 1997 [PMCID: PMC1159851] [PubMed:9379421]

Schuurmans J, De Vlugt E, Schouten AC, Meskers CGM, De Groot JH, Van Der Helm FCT. Themonosynaptic Ia afferent pathway can largely explain the stretch duration effect of the long latency M2response. Exp Brain Res 193: 491–500, 2009 [PubMed: 19048240]

Sinkjaer T, Andersen JB, Nielsen JF, Hansen HJ. Soleus long-latency stretch reflexes during walking inhealthy and spastic humans. Clin Neurophysiol 110: 951–959, 1999 [PubMed: 10400211]

Sinkjaer T, Toft E, Andreassen S, Hornemann BC. Muscle stiffness in human ankle dorsiflexors: intrinsicand reflex components. J Neurophysiol 60: 1110–1121, 1988 [PubMed: 3171659]

Sonnenborg FA, Andersen OK, Arendt-Nielsen L. Modular organization of excitatory and inhibitory reflexreceptive fields elicited by electrical stimulation of the foot sole in man. Clin Neurophysiol 111: 2160–2169, 2000 [PubMed: 11090767]

Suresh NL, Ellis MD, Moore J, Heckman H, Rymer WZ. Excitatory synaptic potentials in spastic humanmotoneurons have short rise-time. Muscle Nerve 32: 99–103, 2005 [PubMed: 15786417]



Thilmann AF, Schwarz M, Töpper R, Fellows SJ, Noth J. Different mechanisms underlie the long-latencystretch reflex response of active human muscle at different joints. J Physiol 444: 631–643, 1991[PMCID: PMC1179953] [PubMed: 1840409]

Türker KS, Cheng HB. Motor-unit firing frequency can be used for the estimation of synaptic potentials inhuman motoneurones. J Neurosci Methods 53: 225–234, 1994 [PubMed: 7823625]

Türker KS, Powers RK. Black box revisited: a technique for estimating postsynaptic potentials in neurons.Trends Neurosci 28: 379–386, 2005 [PubMed: 15927277]

Türker KS, Powers RK. Effects of large excitatory and inhibitory inputs on motoneuron discharge rate andprobability. J Neurophysiol 82: 829–840, 1999 [PubMed: 10444680]

Türker KS, Powers RK. Estimation of postsynaptic potentials in rat hypoglossal motoneurones: insightsfor human work. J Physiol 551: 419–431, 2003 [PMCID: PMC2343211] [PubMed: 12872008]

Türker KS, Schmied A, Cheng HB. Correlated changes in the firing rate of human motor units duringvoluntary contraction. Exp Brain Res 111: 455–464, 1996 [PubMed: 8911940]

Türker KS, Yang J, Brodin P. Conditions for excitatory or inhibitory masseteric reflexes elicited by toothpressure in man. Arch Oral Biol 42: 121–128, 1997 [PubMed: 9134124]

van Doornik J, Kukke S, Sanger T. Hypertonia in childhood secondary dystonia due to cerebral palsy isassociated with reflex muscle activation. Mov Disord 24: 965–971, 2009 [PubMed: 19353733]

Voigt M, De Zee M, Sinkjaer T. A fast servo-controlled hydraulic device for the study of musclemechanical and reflex properties in humans. Book of Abstracts, 17th Congress of the InternationalSociety of Biomechanics, ISB 99, 8–13 August 1999, Calgary, Canada, 1999, p. 578

Yamamoto S, Nakazawa K, Yano H, Ohtsuki T. Differential angle-dependent modulation of the long-latency stretch reflex responses in elbow flexion synergists. J Electromyogr Kinesiol 10: 135–142,2000 [PubMed: 10699561]

Figures and Tables



Fig. 1.

Experimental setup. A schematic view shows the foot-actuator interface and the forces and accelerations measured. Theposition of the adapter was adjusted such that the anatomic axis of the ankle joint of each subject coincided with theturning point of the actuator (TP). The imposed forces were measured with a linear load cell (Slimline; Kistler) attached tothe footplate. The acceleration was measured with an accelerometer (KSHEAR Piezotron; Kistler) attached in line withthe load cell. Amp Ch1 and Amp Ch2, amplifier channels 1 and 2.



Fig. 2.

Joint angle changes and reflex responses. The motor unit responses to 3 different perturbations (tap-like, A; ramp andhold, B; half-sine, C) are shown. Dashed vertical lines indicate the trigger time of perturbation. Top recordings show thechange in joint angle during perturbations; middle and bottom recordings show the cumulative sums (CUSUM) ofperistimulus time histogram (PSTH) and peristimulus frequencygram (PSF) analyses.



Table 1.

Latencies, durations, and amplitudes of significant deflections (reflex responses) in CUSUMs ofSEMG, PSTH and PSF for tap stimulus

Values are means ± SE for reflex responses of motor units to tap stimuli and are presented as cumulative sums(CUSUMs) of surface electromyography (SEMG), peristimulus histograms (PSTH), and peristimulusfrequencygrams (PSF). Amplitudes of reflex responses for SEMG are percentages of the maximal prestimulusdeflection (%max); the sizes of reflex responses for PSTH and PSF are excitatory postsynaptic potential (EPSP)normalized to the number of triggers (extra spike counts per trigger and extra Hz per trigger, respectively). M1, short-latency stretch reflex; M2, second stretch-related reflex; M3, long-latency excitation; S1, first silent period; S2, long-duration silent period.

SEMG PSTH

Latency,ms

Duration,ms

Amplitude,%max

Incidence Latency,ms

Duration,ms

EPSPsize, ext-

count/trig


D

M1 47 ± 3 15 ± 2 73 ± 23 67/72 47 ± 4 11 ± 4 0.241 ±0.091

63/72 50 ± 4 12

M2 76 ± 3 10 ± 3 44 ± 22 23/72 75 ± 3 13 ± 7 0.112 ±0.070

27/72 75 ± 3 55

M3 131 ± 20 42 ± 16 25 ± 9 57/72 140 ± 19 42 ± 15 0.193 ±0.085

49/72 125 ± 27 64

S1 62 ± 3 27 ± 14 39 ± 15 60/72 58 ± 4 31 ± 15 0.166 ±0.090

59/72

S2 89 ± 19 38 ± 19 29 ± 13 38/72 86 ± 9 39 ± 19 0.198 ±0.109

38/72



Fig. 3.

Open in a separate window

Reflex response of a single tibialis anterior (TA) motor unit to the tap-like stretch stimulus applied on the Achilles tendon.Time 0 indicates the timing of the tap-like stretch stimulus (1). The reflex response of the motor unit to 300 stimuli isexpressed using surface electromyography (SEMG) and its CUSUM (top 2 traces), PSTH and its CUSUM (middle 2traces), and PSF and its CUSUM (bottom 2 traces). Horizontal dashed lines indicate maximal CUSUM deflection duringthe 400 ms preceding the stimulus, i.e., the error box for CUSUM. Reasoning for building the error box via examining forthe largest deflection during the prestimulus period (similar period to the minimum reaction time to the stimulus; around200 ms) is detailed by Türker and colleagues (Brinkworth and Türker 2003; Türker et al. 1997), and its validity has beendirectly tested on regularly discharging motoneurons in rat brain slices (Türker and Powers 2003). In accordance with theerror box approach, any poststimulus deflection that is larger than the error box and occurs before the minimum reactiontime to the stimulus is classified as a significant reflex. Vertical dashed lines 2–6 indicate significant reflexes inaccordance with the PSTH-CUSUM, which relies on comparison of the CUSUM error box with that of the largerpoststimulus deflections. Line 2 indicates the initiation of the short-latency stretch reflex, or M1; line 3 indicates the first

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3921411/figure/F3/?report=objectonly



silent period, or SP1; line 4 indicates the second stretch-related reflex, or M2; line 5 indicates the second and long-duration silent period, or SP2; and finally, line 6 indicates the long-latency excitation, or M3. The bin width for SEMGand PSTH was 500 μs; for PSF it was 100 μs. Note that the amplitudes of PSTH and PSF CUSUMs are normalized to thenumber of stimulus events (ext-counts/trigger, extra spike counts per trigger; ext-Hz/trigger, extra Hz per trigger). Thisapproach was essential for comparing the size of the reflex responses because any increase in the number of stimuluspulses would enhance the absolute amplitude of the CUSUM records and because none of the units received an identicalnumber of stimulus pulses. Note also that in response to the stimulus, the SEMG and PSTH indicate 5 separate significantreflexes; the PSF only confirms 2 of the 5 as genuine reflexes because only during M1 and M2 were there significantdischarge rate changes in the unit. During the SPs and M3, on the other hand, the discharge rate of the unit stayed similarto the prestimulus rate.



Fig. 4.


Reflex response of a single TA motor unit to the ramp-and-hold stretch stimulus (n = 204 stimuli). Properties of the traces,horizontal lines, and vertical lines 1–6 are similar to those indicated in Fig. 3. Again, whereas SEMG and PSTH indicatedat least 4 significant reflex responses, PSF only confirmed the 2 excitatory events, M1 and M2, as genuine reflexresponses where the discharge rate of the unit increased significantly.





Table 2.

Latencies, durations, and amplitudes of significant deflections (reflex responses) in CUSUMs ofSEMG, PSTH, and PSF for ramp-and-hold stimulus

Values are means ± SE for reflex responses of motor units to ramp-and-hold stimuli and are presented as CUSUMs ofSEMG, PSTH, and PSF.

SEMG PSTH

Latency,ms

Duration,ms

Amplitude,%max


Duration,ms

EPSPsize, ext-

count/trig


D

M1 48 ± 3 19 ± 4 83 ± 42 15/16 48 ± 4 15 ± 4 0.358 ±0.163

14/16 52 ± 4 13

M2 81 ± 6 20 ± 9 86 ± 52 14/16 80 ± 7 12 ± 3 0.279 ±0.104

11/16 78 ± 6 28

M3 131 ± 17 40 ± 13 35 ± 19 12/16 132 ± 13 32 ± 15 0.311 ±0.117

10/16 129 ± 16 40

S1 67 ± 4 13 ± 2 30 ± 8 8/16 61.0 ± 6 21 ± 12 0.167 ±0.085

5/16

S2 100 ± 10 37 ± 14 42 ± 20 13/16 98 ± 13 42 ± 17 0.306 ±0.254

12/16



Fig. 5.


Reflex response of a single TA motor unit to the sine-type stretch stimulus (n = 283 stimuli) applied on the Achillestendon. Properties of the traces, horizontal lines, and vertical lines 1–3 are similar to those indicated in Fig. 3. There wereonly 2 significant reflex events (LLE, long-lasting excitation; LLSP, long-lasting silent period) in SEMG and PSTH butonly 1 (LLE) in PSF.





Table 3.

Latencies and amplitudes of significant deflections (reflex responses) in CUSUMs of SEMG,PSTH, and PSF for half-sine stimulus

SEMG PSTH PSF

Latency,ms

Amplitude,%max

Latency,ms

EPSP size, ext-count/trig

Latency,ms

EPSP size, ext-Hz/trig

LLE 68 ± 13 36 ± 16 83 ± 10 0.356 ± 0.137 91 ± 11 3.975 ± 2.540

LLSP 145 ± 1 21 ± 2 145 ± 5 0.247 ± 0.090

Values are means ± SE for reflex responses of motor units to half-sine stimuli and are presented as CUSUMs ofSEMG, PSTH, and PSF. LLE, long-lasting excitation; LLSP, long-lasting silent period.



Fig. 6.

Similarities between the reflex responses to the tap-like and ramp-and-hold type stretch stimuli. CUSUMs of the PSF,PSTH, and SEMG are shown for a unit that received first tap-like and then ramp-and-hold-like stretch stimuli (n = 260and 303 stimuli, respectively). The thick vertical dot-dashed line indicates the timing of the stimulus, line 1 indicates thelatency of M1, line 2 indicates the latency of M2, line 3 illustrates the start of the silent period (SP), and line 4 shows thelatency of M3. Note again that whereas 4 separate reflex responses are observed in the SEMG and PSTH, only 2excitatory responses are indicated in the PSF: M1 and M2. Furthermore, although M3 was shown to be an excitatoryevent according to the SEMG and PSTH, the discharge rate of the underlying spikes (see PSF-CUSUM) actuallydecreased during this period.



Fig. 7.


Dependence of the M1 and M2 responses on the background discharge rate of the unit. Three hundred tap-like stimuli at8.6 Hz, 120 at 9.2 Hz, 300 at 10.5 Hz, and 264 at 10.9 Hz induced a common 5 phase reflexes in the SEMG and itsCUSUM. Four different simultaneously active units that fired at differing rates had varying sizes of the M1 and M2 reflexresponses. Note that whereas the M1 response decreased with the increase in the background rate of the unit, theamplitude of the M2 response actually increased along with the rate.

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