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Cutaneous silent period evoked in human first dorsal interosseous muscle motor units by laser stimulation
Journal of Electromyography and Kinesiology 31 (2016) 104–110
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Cutaneous silent period evoked in human first dorsal interosseous muscle motor units by laser stimulation Mehmet Cemal Kahya a,⇑, Og˘uz Sebik b, Kemal S. Türker b a b
_ _ Department of Biophysics, Izmir Katip Çelebi University School of Medicine, Izmir, Turkey Department of Physiology, Koç University School of Medicine, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 28 February 2016 Received in revised form 28 September 2016 Accepted 10 October 2016
Keywords: Nociceptive laser stimulus Peristimulus frequencygram Peristimulus time histogram Reflex inhibition Single motor unit electromyogram Surface electromyogram
a b s t r a c t Painful stimulation of the hand results in an inhibitory response in the hand muscles known as the cutaneous silent period (CSP). In this study, we employed probability- and frequency-based analysis methods to examine the CSP induced by laser stimuli. Subjects were asked to contract their first dorsal interosseous muscle so that selected motor units discharged at a rate of about 8 Hz. Laser pulses were delivered to the palm of the hand, and reflex responses were recorded. The stimuli generated CSP in all test subjects. We found that the latency of the CSP evoked using laser stimulation was longer than that the previously published latency values of the CSP evoked using electrical stimulation. Using only the presently generated laser induced CSP data, the CSP duration was longer when analyzed via peristimulus frequencygram method compared to the probability-based methods such as peristimulus time histogram and surface electromyogram. In the light of the current results, we suggest that laser stimulation could be used when studying pain pathways in human subjects and the frequency-based analysis methods can be preferred because they are previously shown to be more reliable for obtaining the synaptic activity profile. These results can be used to standardize the CSP methods in basic and clinical research. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Painful cutaneous stimulation of human subjects induces reflex inhibition of the ongoing electromyogram (EMG) activity in neighboring muscles (Inghilleri et al., 1997; Kranz et al., 1973; Romaniello et al., 2004; Truini et al., 2009). This reflex is called the cutaneous silent period (CSP), and it is a component of a defensive reflex mechanism that protects the limbs from painful stimuli (Inghilleri et al., 1997; Kofler et al., 2007). The properties of this reflex have been the subject of much clinical interest because it is linked to various sensory neuropathies (Kofler, 2003; Leis, 1994; Tataroglu et al., 2005; Uncini et al., 1991): entrapment neuropathies, such as carpal tunnel syndrome (Aurora et al., 1998; Kofler et al., 2003; Svilpauskaite et al., 2006; Truini et al., 2009), Parkinson’s disease, and dystonia (Pullman et al., 1996; Serrao et al., 2002). For example: Pullman and colleagues have reported prolonged CSPs in hand muscles bilaterally in 11 patients with unilateral hand dystonia, and in 7 patients with Parkinson’s disease (Pullman et al., 1996). Serrao and colleagues _ ⇑ Corresponding author at: Department of Biophysics, Izmir Katip Çelebi _ University Faculty of Medicine, Izmir, Turkey. E-mail address: [email protected] (M.C. Kahya). http://dx.doi.org/10.1016/j.jelekin.2016.10.002 1050-6411/Ó 2016 Elsevier Ltd. All rights reserved.
reported that the prolonged CSPs in 14 patients with Parkinson’s disease were improved with levodopa treatment (Serrao et al., 2002). The afferent limb of the CSP has been estimated to be the A-delta group of myelinated fibers (Inghilleri et al., 1997; Kranz et al., 1973; Kumru et al., 2009; Romaniello et al., 2004). However, some studies have shown that CSP is not supported purely by nociceptive fibers, but instead, its afferents may also have an A-beta component (Serrao et al., 2001). These claims and counter-claims are difficult to substantiate because most previous studies used electrical stimuli to elicit CSP. It is well known that the thickest myelinated fibers (low-threshold fibers, such as A-alpha and Abeta fibers) are activated at low intensities of electrical stimulation, whereas higher-threshold thinner fibers, such as A-delta fibers, are activated at the higher levels of electrical stimulus intensity (Serrao et al., 2001). Thus, the CSP latency, duration, and amplitude obtained using painful electrical stimuli may be affected by the combined activation of both high- and low-threshold sensory nerve fibers, and therefore, they may not be reliable for indicating the afferent limb of the CSP circuitry. Unlike the electrical stimulation of the skin, cutaneous laser stimuli can selectively activate A-delta and C mechano-heat
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nociceptors (Romaniello et al., 2002, 2004), thereby avoiding contamination by the A-alpha and A-beta fiber circuitries that are activated in electrical stimulation methods (Bromm and Treede, 1991). In this study, we attempted to selectively activate nociceptive afferents using laser stimuli in order to evoke CSP mediated by A delta afferents. We hypothesized that the laser-induced CSP has a longer reflex latency compared with electrically-induced CSP. The rationale for this hypothesis is as follows: Since the electrical stimulation must have activated the low threshold A-alpha and -beta fibers as well as the high threshold A-delta fibers to elicit CSP, the latency for the electrically-induced CSP may be affected by the fast conducting A-alpha and beta fibers and hence would be shorter compared with the laser induced CSP. This is due to the fact that the laser stimulation preferentially activates slower conducting A-delta fibers in order to evoke CSP. Other reflex properties may also change as the two forms of stimuli use different afferents to elicit the reflex. We also investigated the properties of CSP using two different analysis methods: discharge rate-based and classical probabilitybased analysis methods. Using known postsynaptic potentials (PSPs) on regularly discharging motor neurons in rat brain slices it is established that the PSF method is a more useful method for indicating the total duration and the profile of the underlying PSP than the classical methods such as the peristimulus time histogram (PSTH) and surface EMG (SEMG). Therefore, we hypothesize that the discharge rate-based peristimulus frequencygram (PSF) method can estimate the CSP profile which is similar to the actual PSP in the postsynaptic cell compared with the PSP profile elicited using the probability-based methods, such as PSTH and SEMG. 2. Methods 2.1. Subjects Ten right-handed, neurologically healthy adult volunteers (four men and six women) participated in the experiments. The mean age of the subjects was 30 ± 2.4 years. Written informed consent was obtained from each subject. All experimental procedures were approved by the Ethics Committee for Human Experimentation at Izmir Katip Celebi University (Protocol number: 07.03.2013-28), and they were performed in accordance with the principles of the Declaration of Helsinki. 2.2. Experimental protocol All data were recorded with the subject sitting in an upright position on a comfortable dental chair with the elbow at 90 degrees of flexion. The right hand was held in a pronated position with the fingers straight. The thumb was held at 45 degrees of abduction on a hand support (Fig. 1). The subject was instructed to abduct his/her index finger so the first dorsal interosseous muscle (FDI) was activated. The single motor unit activity of the FDI was recorded using custom-made intramuscular fine wire bipolar electrodes, which were produced from a pair of TeflonÒ-coated silver wires. The wires were insulated except for the tips in order to record the activity of a single unit. Using 25 G needles, the electrodes were placed into the belly of the FDI muscle at a depth of approximately 1 cm. The needle was retracted following insertion to leave the wires in the muscle. The subjects were grounded using a lip-clip electrode (Türker et al., 1988). Single-unit discharges were bandpass filtered in the range of 200–10,000 Hz, sampled at 20 kHz, recorded using a CED system comprising a CED 1902 and a CED Power1401 (Cambridge Electronic Design Limited, Cambridge,
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Fig. 1. Hand holder: The right hand of the subject was held in a restricted position to isolate the activity of the FDI muscle. The positions of the surface and intramuscular electrodes on the FDI muscle are also indicated.
UK), and transferred to a computer using the CED Spike2Ò data acquisition program (Cambridge Electronic Design Limited, Cambridge, UK). The SEMG was obtained from the muscle by placing bipolar electrodes 2 cm apart on the muscle belly (Fig. 1). In order to reduce electrode resistance to <5 kX, the skin where we placed the electrodes was cleaned using 70% alcohol. The SEMG signals were amplified 1000 times and band-pass filtered with cut-offs at 20 and 500 Hz. The signals were sampled at 2 kHz and recorded using the CED system. The data were analyzed using the CED Spike 2Ò program (Cambridge Electronic Design Limited, Cambridge, UK). Subjects were asked to contract their muscles until one or two motor units were recruited on the single-unit electrode. They were provided with auditory feedback about the activity of the most prominent unit (i.e., the one for which the largest motor unit action potential was being recorded). Using the auditory feedback, the subjects were asked to keep the motor unit discharging at a rate of approximately 8 Hz. The reflex responses were recorded using both SEMG and intramuscular EMG while the subject kept the selected motor unit discharging at a constant rate. During the discharge of the selected motor unit, the SEMG levels stayed always below the 5% of the maximal SEMG recorded for that experiment. This low contraction level was similar in our earlier paper in which electrical stimulation was used to study CSP in the same muscle (Kahya et al., 2010). 2.3. Stimulation A laser beam was targeted on the C8 dermatome on the palm of the hand using a CO2-laser stimulator (Neurolas, Electronic Engineering, Florence, Italy). The laser pulses (wavelength = 10.6 lm, intensity = 1.5–15 W, duration = 15 ms) had a beam diameter of 2 mm when irradiating an area of about 3 mm2, with a duration of 15 ms, and a beam diameter of 4 mm when irradiating an area of about 13 mm2. The laser beam was moved slightly to cover the area shown in Fig. 2 (about 10 cm2) after each stimulus to avoid fatigue or sensitization in the nociceptors, where the stimuli were triggered randomly by a computer at intervals of between 5 and 10 s. The cutaneous innervations that supply the C8 myotome, which includes the FDI muscle, are present in the C8 dermatome. The stimulus intensity was increased incrementally up to a level where consistent periods of inhibition were readily observable in the SEMG traces. At least 120 stimuli were used for each trial in the experiments to test 12 motor units. Participants scored the stimuli on an 11-point visual analog scale (VAS) at the end
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2.5. Determination of reflexes The procedure for identifying latency and duration of reflexes was fully described in our earlier paper (Kahya et al., 2010). Briefly, we have determined the prestimulus variations on the CUSUM record to establish the error box (Türker and Powers, 2005; Brinkworth and Türker, 2003). Any post stimulus deflection that was larger than the error box and appeared before the reaction time to the laser pulse stimulus was considered as a significant reflex response to the stimulus. The onset of CSP was marked at the point where the first significant downward deflection occurred in SEMG-CUSUM or PSTH-CUSUM traces. Significant upturn on the other hand is considered to be reflecting the end of the inhibitory period in the probability based analyses. PSF analysis on the other hand uses the same procedure as the PSTH-CUSUM for the onset of the reflex (see the Discussion for the reasons underlying this procedure). The end point of the inhibitory period in the PSF is when the discharge rate of the motor unit returns to the prestimulus levels. Fig. 2. Area for laser stimulation: Laser stimulation was applied to the area of the C8 dermatome on the palmar side (hatched area) and laser handle.
of each trial where 0 indicated no pain and 10 indicated unbearable pain.
2.4. Analyses The SEMG recordings were full-wave rectified and averaged in a 1000 ms window centered on the stimulus. Using the Spike2Ò (CED) program, single motor units were identified and marked using a template matching algorithm. Based on the discharge times of individual motor units, acceptance pulses were created by the Spike2 program and placed into 1-ms bins around the time of the stimulus to construct the PSTH. The PSF is formed by the superimposition of the instantaneous discharge rates of a single motor unit in the temporal neighborhood of the stimuli, where PSF indicates the excitability of the motoneuron membrane (Türker and Cheng, 1994). Thus, the PSFs were constructed using the acceptance pulses obtained from the Spike2 program, and the instantaneous discharge rate values were calculated by taking the multiplicative inverse of the interspike intervals (in seconds). The instantaneous discharge rate values were then placed at the position of the discharge time of the latter spike in an interspike interval. The PSFs were obtained by superimposing the calculated instantaneous discharge rate values around the time of the stimulus using at least 120 stimuli. Cumulative sums (CUSUM; Ellaway, 1978) were obtained using the averaged SEMG to determine the exact timing of the reflex response. CUSUMs were also calculated for PSTH and PSF graphs to detect any subtle but significant changes (Kahya et al., 2010).
2.6. Statistics After confirming the existence of the reflex response using the criteria defined above, we compared the latency and duration values to detect differences between the values calculated with SEMG and PSTH versus PSF methods using Wilcoxon’s signed rank test, where p < 0.05 was considered as significant (Table 1). We also compared the CSP results with our previous results (Kahya et al., 2010) for electrically-induced CSP using Wilcoxon’s signed rank test. 3. Results We obtained 17 SEMG records from 10 subjects and using these 17 records we analyzed results for 12 motor units. This was due to the fact that a minimum of 120 stimuli are needed to obtain reliable single unit analyses and some units disappeared before this stimulus number was reached. According to our experience, large numbers of triggers are needed so that there are sufficient spikes to reliably indicate the changes in the discharge probability and rate around the time of the stimulus (Binboga and Türker, 2012; Yavuz et al., 2014). Significant CSPs were observed in all 17 SEMG recordings. The stimulus intensity that induced CSPs was rated by the participants as between 5 and 6 (mean = 5.46) based on a VAS of 0–10. The mean perception threshold was 65.8 ± 6.5 mJ. The stimulus intensity required to observe CSP was 3–10 times the perception threshold (207.7 ± 12 mJ). The reflex responses were evaluated simultaneously using SEMG, PSTH, and PSF analysis methods, as shown by the example in Fig. 3. The latency of CSP current study was 72 ms for the SEMGCUSUM; and 67 ms for the PSTH-CUSUM. The endpoint values for CSP determined using the probabilistic methods (SEMG and PSTH)
Table 1 Comparison of the latencies and durations of CSP. SEMG recordings and simultaneously recorded single motor units are compared with the values derived from the CUSUMs of SEMG, PSTH, and PSF. SEMG (17 records) Latency of CSP Endpoint of CSP Duration of CSP
72.47 (3.04) 146.59 (7.11) 74.12 (6.16)
PSTH (12 motor units) 67.08 (5.07) 136.08 (6.66) 69.00 (5.79)
PSF (12 motor units) a
67.08 (5.07) 201.67 (7.26)b 134.50 (8.59)
SEMG vs. PSTH p-value*
SEMG vs. PSF p-value*
PSTH vs. PSFa p-value*
0.074 0.113 0.722
0.010 0.010 0.423
– 0.002 0.002
Results represent the mean (ms) ± standard errors shown in brackets. n = motor unit numbers. * Wilcoxon’s signed rank test. a Latency calculations were based on values obtained by the PSTH method. b Endpoint calculations were determined based on the PSF method.
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Fig. 3. Simultaneously recorded reflex responses using two probability-based methods (SEMG and PSTH) and one frequency-based (PSF) analysis methods: Rectified + averaged SEMG and the CUSUM are displayed in the top trace, PSTH and the CUSUM in the middle, and PSF in the bottom trace. From 1 to 4, the vertical lines indicate: (1) position of the stimulus at time zero; (2) latency of the CSP based on the PSTH-CUSUM; (3) end of CSP as indicated by SEMG-CUSUM; and (4) latency of the ‘‘excitatory” period as indicated by SEMG-CUSUM. The endpoint of CSP in PSTHCUSUM and the starting point of CSP in PSF-CUSUM are indicated by downward arrows. The average discharge rate in the PSF-CUSUM record is shown as a solid trace (obtained using moving average of the bin values for 5 ms). The maximum variation in the average discharge rate during the 100-ms prestimulus period is indicated by broken horizontal lines (error box). The y-axis of the PSTH chart shows the number discharges of the motor unit in a given time bin.
were significantly different compared with the values calculated using the frequency-based method (PSF). The duration of CSP was significantly longer when calculated using the PSF method compared with that calculated using the SEMG method (p = 0.002) (Table 1). A typical experiment (original records of single motor units and SEMG) with all of the analyzed variables is illustrated in Fig. 3, where vertical line ‘‘2” indicates the beginning of CSP and vertical line ‘‘3” indicates the termination of CSP determined using SEMGCUSUM. These points were marked using downward vertical arrows in PSTH-CUSUM and PSF average frequency traces. The CSP duration, which was determined as the period between lines 2 and 3 in SEMG-CUSUM, was longer compared with the CSP duration calculated using PSTH-CUSUM, but it was shorter than the CSP duration calculated using PSF. We observed reduced firing frequency values in the PSF graph during inhibition, as shown in Figs. 3 and 4.
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Fig. 4. PSTH, PSTH-CUSUM, and PSF for a single unit from a different subject. The explanations are similar to those given for Fig. 3, where the vertical lines from 1 to 4 indicate the following: (1) position of the stimulus at time zero; (2) latency of CSP based on PSTH-CUSUM; (3) end of CSP as indicated by SEMG-CUSUM; and (4) latency of the ‘‘excitatory” period as indicated by SEMG-CUSUM. The endpoint of CSP in PSTH-CUSUM and the starting point of CSP are indicated by arrows in the average PSF trace. The average discharge rate in the PSF record is shown as a solid trace (obtained using moving average of the bin values for 5 ms). It should be noted that the discharge rate values were lowest in PSF during the ‘‘excitatory” peak in PSTH. The maximum variation in the average discharge rate during the 100-ms prestimulus period is indicated by broken horizontal lines (error box). The y-axis of the PSTH chart shows the number discharges of the motor unit in a given time bin.
In Fig. 4, the differences between SEMG, PSTH, and PSF methods in terms of the CSP duration are further illustrated using a single motor unit from a different subject. Similar points to those described above can also be observed in this figure. Table 2 summarizes our CSP results with those obtained in other studies using both electrical and laser stimulation. 4. Discussion In this study, we obtained several important new results. First, laser stimulation of the hand induced CSP in all of the FDI motor units tested. Second, the duration of the CSP differed according to the analysis program employed. Third, the latency of the CSP elicited using laser stimulation differed from the CSP latency values reported in earlier publications that used electrical stimuli.
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Table 2 CSP in FDI: comparison of current results (bold numbers) and those obtained in other studies (light numbers).
a b
Stimulation site
Latency, ms Mean (SD)
Duration, ms Mean (SD)
Subjects (n)
Authors
Electrical stimulation
Palmar side of the hand (palmar C-8) Digit V (C8 dermatome) Dorsal side of the hand (dorsal C-8)
65 (7) 69.4 (4.34) 58 (2.8) 63.7 (2.8) 70.8 (3.7)
54 (6) 46.7 (6.79) 71.6 (8.1) 74.0 (6.6) 124.5 (11.7)
5 7 6 (18 units) 6 6 (18 units)
Romaniello et al. (2004) Inghilleri et al. (2002) Kahya et al. (2010) (probabilistic-PSTH-CUSUM) Kahya et al. (2010) (probabilistic-SEMG-CUSUM) Kahya et al. (2010) (PSF)
Laser stimulation
Palmar side of the hand (palmar C-8)
90 (7) 67.08 (5.07) 72.47 (3.04) 67.08 (5.07)b
40 (6) 69.00 (5.79) 74.12 (6.16) 134.50 (8.59)
5 10 (12 units) 10 (17 records)a 10 (12 units)
Romaniello et al. (2004) Current results (probabilistic-PSTH-CUSUM) Current results (probabilistic-SEMG-CUSUM) Current results (PSF)
Results were obtained based on 17 SEMG recordings obtained from 10 subjects. Latency calculations were based on values obtained using the PSTH method.
4.1. CSP latency and duration obtained from probability and discharge rate analyses We used probability- and discharge rate-based analyses to investigate the properties of CSP in human FDI muscle induced by painful laser stimuli. In these analyses, the PSF can only provide limited information regarding the onset of an IPSP due to the limited number of discharges during the inhibitory period; therefore, we used the disappearance of the spikes in PSTH-CUSUM to determine the latency of the reflex responses of single motor units for both PSF and PSTH calculations. This approach was explained in detail by Todd et al. (2012) and Ugincˇius et al. (2014). This procedure was also demonstrated to be appropriate for determining IPSP latency in our previous brain slice experiments, where we studied the effect of IPSPs and excitatory postsynaptic potentials (EPSPs) on regularly discharging motor neurons in brain slices (Türker and Powers, 1999). The latency of CSP induced using electrical stimulation in our previous study (Kahya et al., 2010) using the same method was significantly shorter than the latency of CSP in the current study (probabilistic method (SEMG-CUSUM): 63.7 versus 72.5 ms; p < 0.05). In contrast, the termination of CSP is not easily determined via the probability-based classical methods, i.e., SEMG or PSTH. In the classical methods, the termination of the inhibitory period is marked at the time point when a significant activity brings SEMG or bin counts in PSTH back to the prestimulus level or higher following a trough. In the PSF approach, however, the termination of the inhibitory period is determined when the average discharge rate trace returns to the mean prestimulus discharge rate line (Türker and Powers, 1999). This approach was also tested successfully in our regularly discharging rat motor neuron experiments (Türker and Powers, 1999). Fig. 3 and Tables 1 and 2 show that there was a reduction in the discharge rate following the laser stimuli, which had its endpoint 201 ms after the time of the stimulus (time zero). When calculated using the PSF data, the duration of CSP was as long as 134 ms, whereas the CSP duration value calculated using probabilistic methods was 69 ms (also see Romaniello et al., 2004). Depending on the method used, there was a difference of about 65 ms in the calculated CSP durations. The discrepancy between the two calculated values was due to the methodological shortcomings of the probability-based methods when evaluating the duration of inhibitory inputs. We have shown in motoneurons in brain slices that a simple IPSP injected into a regularly discharging motoneuron generates four peaks and troughs in the probability-based analyses such as PSTH. Immediately after the delivery of an IPSP, PSTH indicates a trough followed by a peak in the probability of the spike numbers. Therefore, a peak in the histogram occurs during the rising phase of the injected IPSP (please also refer to Figs. 7 and 8 in Türker and Powers, 1999) and hence places a limit on the duration
of the genuine inhibitory period. SEMG analysis also has similar shortcomings as detailed in Türker and Powers (2003). The PSF analysis on the other hand follows the shape of the IPSP without generating an error in estimating the actual duration of the injected IPSP. Therefore, we are proposing in the current study that the genuine duration of the CSP can be estimated using the PSF method as the PSTH and SEMG methods induce a peak while the IPSP is still continuing and hence underestimate its duration significantly. Furthermore, PSTHs contain secondary peaks and troughs, which are not related directly to the underlying PSP, but instead, they reflect the regular recurrence of spikes (autocorrelogram) following those affected by PSP (Türker and Powers, 1999). PSF analysis is more useful for indicating the total duration and the profile of the underlying PSP. The shape of the underlying PSP can be obtained directly from the PSF records because the discharge rates of the spikes follow the PSPs very closely (Türker and Powers, 1999). The PSF method has been shown to provide a more accurate evaluation of these inputs using known inputs based on regularly discharging motor neurons in rat brain slices (please refer to Figs. 7 and 8 in Türker and Powers, 1999). According to our findings, it is evident that classical knowledge regarding the CSP duration must be revised. A similar result was found for the CSP evoked using electrical stimulation, and its possible implications were discussed by Kahya et al. (2010).
4.2. Laser versus electrical stimulation for inducing the CSP High and low threshold nerve fibers are activated by strong electrical stimuli (Staahl and Drewes, 2004), and thus it would be ideal to use specific stimulation techniques where only the highthreshold fibers are activated to examine the neuronal networks underlying pain pathways responsible for CSP (Plaghki and Mouraux, 2005). Nociceptive laser stimulation of the palm effectively evokes reflex inhibition of the hand muscles, whereas stimulation of the dorsum does not (Romaniello et al., 2004). Laser stimuli activate both myelinated A-delta mechano-heat nociceptors (AMH) and unmyelinated C mechano-heat nociceptors (CMH), but it is not possible to measure the possible contribution of CMH units to the evocation of CSP because the conduction velocity of these unmyelinated fibers is too slow (Romaniello et al., 2004). Type I AMH units require a long-lasting heat stimulus, and the response has a significant delay (in the order of seconds). By contrast, the response of the type II AMH units has a short latency when a short-lasting heat (laser) stimulus is used (Treede et al., 1998). In contrast to type II AMH units, type I AMH units have a higher conduction velocity and lower mechanical thresholds. Thus, due to their corresponding receptor characteristics, it may be expected that AMH I units will respond first to mechanically-
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induced pain, whereas AMH II units will respond first to heatinduced pain (Treede et al., 1995). Laser-induced heat alone is considered to be insufficient to induce an AMH I response. However, the rapid evaporation of water from the outer layers of skin following the laser pulse may act as a mechanical stimulus to activate AMH I units given the low mechanical threshold for these units (Treede et al., 1998). Therefore, the overall effect of heat and the effective mechanical stimuli created by the rapid increase in temperature could be sufficient to excite AMH I units. Electrical stimuli have several advantages because they are easily applicable, controllable, measurable, and safe (Wells et al., 2007). However, they also have disadvantages such as an electrode touching the tissue and tissue impedance attenuating the amplitude of the stimulus current (Geddes and Roeder, 2003). Moreover, the changes in the stimulation threshold depend on the diameter of the fiber, and nerves may be stimulated even before the stimulus reaches the receptors, which might evoke a response that is not unique to the stimulus (Staahl and Drewes, 2004). Thus, an artifact of the stimulus occurs in many records. Moreover, many records contain noise because of the electric fields generated by the stimulus in the area of interest, especially when small amplitude potentials are recorded. By contrast, stimulation using laser beams does not have the disadvantages mentioned above (Plaghki and Mouraux, 2005). However, the effect of the laser stimulus can differ among each subject due to inter-individual variation in factors such as heat absorption, heat loss, and heat diffusion in the skin (Cruccu et al., 2003; Staahl and Drewes, 2004). There is no defined standard for electrical stimulation, but similar results have been obtained for the CSP latency and duration using classical methods for the FDI muscle (Inghilleri et al., 2002; Kofler, 2003; Romaniello et al., 2004) (Table 2). As shown in Table 2, the CSP latency is 63.7 ms using electrical stimulation (Kahya et al., 2010), whereas it is 72.47 ms when laser stimulation is applied (SEMG-CUSUM). During laser stimulation, there is a lag of 8.77ms in CSP compared with electric stimulation (p = 0.039), possibly because electric stimulation of CSP is generated by both A-beta and A-delta fibers (Bromm and Treede, 1991; Treede et al., 1998, 1995), whereas it is induced by stimulation of the A-delta fibers alone during laser stimulation. Therefore, the difference in the CSP delays may be attributable to differences in the conduction time to the spinal cord and/or the number of interneurons in the circuitry. However, part of this difference in the latency may be attributable to differences in the effective stimulus onset time. An electrical stimulus is instantly effective, but the laser stimulus must heat the skin to activate the nociceptors. The exact time required for stimulation of the pain fibers by the laser pulses is difficult to predict, and this must be considered when evaluating the results of this and similar studies using laser stimuli. The electrical and laser experiments were performed at low contraction levels where only low-threshold units were recruited. In both cases, the contraction levels were below 5% of the maximum voluntary contraction and thus similar low-threshold units may be responsible for the inhibition. 4.3. Clinical application The CSP has been utilized in a number of pathological conditions and in a range of experimental settings to assess the conduction of neuronal pathways in patients (Kofler, 2003; Uncini et al., 1991). Furthermore, CSP has been utilized in patients with numerous sensory neuropathies (i.e., Friedreich’s ataxia, abetalipoproteinemia, and Fabry disease). In addition, CSP has been employed to study entrapment neuropathies, including carpal tunnel syndrome (de Tommaso et al., 2009; Kofler et al., 2003; Svilpauskaite et al., 2006). The CSP has been utilized in studies of
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these neurological disorders because changes in CSP may be related to large diameter fiber impairments. For example, delayed CSP latencies have been reported in a small number of patients with neuropathies that involved large fibers (Floeter, 2003; Uncini et al., 1991). However, CSP was always obtained in the absence of fast conducting sensory nerve fibers in the affected nerve; thus, it has been suggested that the A-delta fibers may be responsible for CSP, so studying CSP may provide information about the stability of the A-delta fibers within a nerve. However, as argued in the present study, laser stimulation is the preferred method that can reliably activate A-delta fibers without involving the thicker myelinated fibers. Hence, we suggest that laser stimulation is used when studying nociceptive fiber pathways in basic and clinical research. As shown by the large number of previous studies in this area, researchers have examined the properties of CSP to obtain data about various pathological conditions for both diagnostic purposes and to follow up the progress or regress of these conditions. Standardizing CSP profile using both probabilistic- and frequencybased analysis methods will be crucial for ensuring that this reflex can be used in clinical settings with more confidence. Author contributions KST and MCK designed the experiment. MCK and OS performed the experiments. KST, MCK, and OS analyzed and interpreted the data. KST and MCK drafted the manuscript and all of the authors approved the final version. The experiments were performed at Koç University. Conflict of interest The authors declare that there are no conflict of interests. Acknowledgements We thank all of the subjects who volunteered for this study. Funding: This research was funded by an internal grant from Koç University, Istanbul, Turkey. References Aurora, S.K., Ahmad, B.K., Aurora, T.K., 1998. Silent period abnormalities in carpal tunnel syndrome. Muscle Nerve 21, 1213–1215. Binboga, E., Türker, K.S., 2012. Compound group I excitatory input is differentially distributed to human soleus motoneurons. Clin. Neurophysiol. 123, 2192–2199. Brinkworth, R.S., Türker, K.S., 2003. A method for quantifying reflex responses from intra-muscular and surface electromyogram. J. Neurosci. Methods 122, 179– 193. Bromm, B., Treede, R.D., 1991. Laser-evoked cerebral potentials in the assessment of cutaneous pain sensitivity in normal subjects and patients. Rev. Neurol. (Paris) 147, 625–643. Cruccu, G., Pennisi, E., Truini, A., Iannetti, G.D., Romaniello, A., Le Pera, D., et al., 2003. Unmyelinated trigeminal pathways as assessed by laser stimuli in humans. Brain 126, 2246–2256. de Tommaso, M., Libro, G., Difruscolo, O., Sardaro, M., Serpino, C., Calabrese, R., et al., 2009. Laser evoked potentials in carpal tunnel syndrome. Clin. Neurophysiol. 120, 353–359. Ellaway, P.H., 1978. Cumulative sum technique and its application to the analysis of peristimulus time histograms. Electroencephalogr. Clin. Neurophysiol. 45, 302– 304. Floeter, M.K., 2003. Cutaneous silent periods. Muscle Nerve 28, 391–401. Geddes, L.A., Roeder, R., 2003. Criteria for the selection of materials for implanted electrodes. Ann. Biomed. Eng. 31, 879–890. Inghilleri, M., Conte, A., Frasca, V., Berardelli, A., Manfredi, M., Cruccu, G., 2002. Is the cutaneous silent period an opiate-sensitive nociceptive reflex? Muscle Nerve 25, 695–699. Inghilleri, M., Cruccu, G., Argenta, M., Polidori, L., Manfredi, M., 1997. Silent period in upper limb muscles after noxious cutaneous stimulation in man. Electroencephalogr. Clin. Neurophysiol. 105, 109–115. Kahya, M.C., Yavuz, S.U., Türker, K.S., 2010. Cutaneous silent period in human FDI motor units. Exp. Brain Res. 205, 455–463.
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Uncini, A., Kujirai, T., Gluck, B., Pullman, S., 1991. Silent period induced by cutaneous stimulation. Electroencephalogr. Clin. Neurophysiol. 81, 344–352. Wells, J., Konrad, P., Kao, C., Jansen, E.D., Mahadevan-Jansen, A., 2007. Pulsed laser versus electrical energy for peripheral nerve stimulation. J. Neurosci. Methods 163, 326–337. Yavuz, S.U., Mrachacz-Kersting, N., Sebik, O., Berna Unver, M., Farina, D., Turker, K.S., 2014. Human stretch reflex pathways reexamined. J. Neurophysiol. 111, 602– 612.
Mehmet C. Kahya is currently working as an Assistant Professor in the department of Biophysics in the Medical Faculty of Izmir Katip Celebi University where he joined in 2011. Prior to that, he worked as a Medical Doctor in many government organizations. His academic qualification includes a degree from Ege University Medical Faculty in 1989. In 2003, he completed General Surgery residency training and in 2010, he received his biophysics PhD from Ege University. His research area includes cell biophysics, biomechanics, bioelectricity, electrophysiology, electromyography and nociceptive reflex response.
Og˘uz Sebik was born in Izmir, Turkey. He received his bachelor’s degree in Physics in 1998 from Koç University and his master’s degree in psychology in 2000 from University of New Haven. He received his Ph.D. from Ege University in Biophysics in 2012. He is currently employed as a post-doctoral researcher at Koç University, School of Medicine. His current research interests include the investigation of the neuronal pathways involved in motor control, and development of new methodologies to help elucidate the intricacies of motor control in humans.
Professor Kemal S. Türker is a dentist and obtained his Ph.D. degree in Physiology at the Glasgow University, Scotland. He took up a research position in the Medical School of Adelaide University, Australia where he worked from 1983 to 2007. He has then returned to his home country, Turkey as the Marie Curie Chair of the European Union. Kemal has devoted all of his efforts towards understanding the synaptic inputs from receptors to motoneurons that innervate human muscles. Current projects vary from control of human mastication by perioral receptors to modulation of various reflexes during movement.
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Journal of Electromyography and Kinesiology journal homepage: www.elsevier.com/locate/jelekin
Cutaneous silent period evoked in human first dorsal interosseous muscle motor units by laser stimulation Mehmet Cemal Kahya a,⇑, Og˘uz Sebik b, Kemal S. Türker b a b
_ _ Department of Biophysics, Izmir Katip Çelebi University School of Medicine, Izmir, Turkey Department of Physiology, Koç University School of Medicine, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 28 February 2016 Received in revised form 28 September 2016 Accepted 10 October 2016
Keywords: Nociceptive laser stimulus Peristimulus frequencygram Peristimulus time histogram Reflex inhibition Single motor unit electromyogram Surface electromyogram
a b s t r a c t Painful stimulation of the hand results in an inhibitory response in the hand muscles known as the cutaneous silent period (CSP). In this study, we employed probability- and frequency-based analysis methods to examine the CSP induced by laser stimuli. Subjects were asked to contract their first dorsal interosseous muscle so that selected motor units discharged at a rate of about 8 Hz. Laser pulses were delivered to the palm of the hand, and reflex responses were recorded. The stimuli generated CSP in all test subjects. We found that the latency of the CSP evoked using laser stimulation was longer than that the previously published latency values of the CSP evoked using electrical stimulation. Using only the presently generated laser induced CSP data, the CSP duration was longer when analyzed via peristimulus frequencygram method compared to the probability-based methods such as peristimulus time histogram and surface electromyogram. In the light of the current results, we suggest that laser stimulation could be used when studying pain pathways in human subjects and the frequency-based analysis methods can be preferred because they are previously shown to be more reliable for obtaining the synaptic activity profile. These results can be used to standardize the CSP methods in basic and clinical research. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Painful cutaneous stimulation of human subjects induces reflex inhibition of the ongoing electromyogram (EMG) activity in neighboring muscles (Inghilleri et al., 1997; Kranz et al., 1973; Romaniello et al., 2004; Truini et al., 2009). This reflex is called the cutaneous silent period (CSP), and it is a component of a defensive reflex mechanism that protects the limbs from painful stimuli (Inghilleri et al., 1997; Kofler et al., 2007). The properties of this reflex have been the subject of much clinical interest because it is linked to various sensory neuropathies (Kofler, 2003; Leis, 1994; Tataroglu et al., 2005; Uncini et al., 1991): entrapment neuropathies, such as carpal tunnel syndrome (Aurora et al., 1998; Kofler et al., 2003; Svilpauskaite et al., 2006; Truini et al., 2009), Parkinson’s disease, and dystonia (Pullman et al., 1996; Serrao et al., 2002). For example: Pullman and colleagues have reported prolonged CSPs in hand muscles bilaterally in 11 patients with unilateral hand dystonia, and in 7 patients with Parkinson’s disease (Pullman et al., 1996). Serrao and colleagues _ ⇑ Corresponding author at: Department of Biophysics, Izmir Katip Çelebi _ University Faculty of Medicine, Izmir, Turkey. E-mail address: [email protected] (M.C. Kahya). http://dx.doi.org/10.1016/j.jelekin.2016.10.002 1050-6411/Ó 2016 Elsevier Ltd. All rights reserved.
reported that the prolonged CSPs in 14 patients with Parkinson’s disease were improved with levodopa treatment (Serrao et al., 2002). The afferent limb of the CSP has been estimated to be the A-delta group of myelinated fibers (Inghilleri et al., 1997; Kranz et al., 1973; Kumru et al., 2009; Romaniello et al., 2004). However, some studies have shown that CSP is not supported purely by nociceptive fibers, but instead, its afferents may also have an A-beta component (Serrao et al., 2001). These claims and counter-claims are difficult to substantiate because most previous studies used electrical stimuli to elicit CSP. It is well known that the thickest myelinated fibers (low-threshold fibers, such as A-alpha and Abeta fibers) are activated at low intensities of electrical stimulation, whereas higher-threshold thinner fibers, such as A-delta fibers, are activated at the higher levels of electrical stimulus intensity (Serrao et al., 2001). Thus, the CSP latency, duration, and amplitude obtained using painful electrical stimuli may be affected by the combined activation of both high- and low-threshold sensory nerve fibers, and therefore, they may not be reliable for indicating the afferent limb of the CSP circuitry. Unlike the electrical stimulation of the skin, cutaneous laser stimuli can selectively activate A-delta and C mechano-heat
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nociceptors (Romaniello et al., 2002, 2004), thereby avoiding contamination by the A-alpha and A-beta fiber circuitries that are activated in electrical stimulation methods (Bromm and Treede, 1991). In this study, we attempted to selectively activate nociceptive afferents using laser stimuli in order to evoke CSP mediated by A delta afferents. We hypothesized that the laser-induced CSP has a longer reflex latency compared with electrically-induced CSP. The rationale for this hypothesis is as follows: Since the electrical stimulation must have activated the low threshold A-alpha and -beta fibers as well as the high threshold A-delta fibers to elicit CSP, the latency for the electrically-induced CSP may be affected by the fast conducting A-alpha and beta fibers and hence would be shorter compared with the laser induced CSP. This is due to the fact that the laser stimulation preferentially activates slower conducting A-delta fibers in order to evoke CSP. Other reflex properties may also change as the two forms of stimuli use different afferents to elicit the reflex. We also investigated the properties of CSP using two different analysis methods: discharge rate-based and classical probabilitybased analysis methods. Using known postsynaptic potentials (PSPs) on regularly discharging motor neurons in rat brain slices it is established that the PSF method is a more useful method for indicating the total duration and the profile of the underlying PSP than the classical methods such as the peristimulus time histogram (PSTH) and surface EMG (SEMG). Therefore, we hypothesize that the discharge rate-based peristimulus frequencygram (PSF) method can estimate the CSP profile which is similar to the actual PSP in the postsynaptic cell compared with the PSP profile elicited using the probability-based methods, such as PSTH and SEMG. 2. Methods 2.1. Subjects Ten right-handed, neurologically healthy adult volunteers (four men and six women) participated in the experiments. The mean age of the subjects was 30 ± 2.4 years. Written informed consent was obtained from each subject. All experimental procedures were approved by the Ethics Committee for Human Experimentation at Izmir Katip Celebi University (Protocol number: 07.03.2013-28), and they were performed in accordance with the principles of the Declaration of Helsinki. 2.2. Experimental protocol All data were recorded with the subject sitting in an upright position on a comfortable dental chair with the elbow at 90 degrees of flexion. The right hand was held in a pronated position with the fingers straight. The thumb was held at 45 degrees of abduction on a hand support (Fig. 1). The subject was instructed to abduct his/her index finger so the first dorsal interosseous muscle (FDI) was activated. The single motor unit activity of the FDI was recorded using custom-made intramuscular fine wire bipolar electrodes, which were produced from a pair of TeflonÒ-coated silver wires. The wires were insulated except for the tips in order to record the activity of a single unit. Using 25 G needles, the electrodes were placed into the belly of the FDI muscle at a depth of approximately 1 cm. The needle was retracted following insertion to leave the wires in the muscle. The subjects were grounded using a lip-clip electrode (Türker et al., 1988). Single-unit discharges were bandpass filtered in the range of 200–10,000 Hz, sampled at 20 kHz, recorded using a CED system comprising a CED 1902 and a CED Power1401 (Cambridge Electronic Design Limited, Cambridge,
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Fig. 1. Hand holder: The right hand of the subject was held in a restricted position to isolate the activity of the FDI muscle. The positions of the surface and intramuscular electrodes on the FDI muscle are also indicated.
UK), and transferred to a computer using the CED Spike2Ò data acquisition program (Cambridge Electronic Design Limited, Cambridge, UK). The SEMG was obtained from the muscle by placing bipolar electrodes 2 cm apart on the muscle belly (Fig. 1). In order to reduce electrode resistance to <5 kX, the skin where we placed the electrodes was cleaned using 70% alcohol. The SEMG signals were amplified 1000 times and band-pass filtered with cut-offs at 20 and 500 Hz. The signals were sampled at 2 kHz and recorded using the CED system. The data were analyzed using the CED Spike 2Ò program (Cambridge Electronic Design Limited, Cambridge, UK). Subjects were asked to contract their muscles until one or two motor units were recruited on the single-unit electrode. They were provided with auditory feedback about the activity of the most prominent unit (i.e., the one for which the largest motor unit action potential was being recorded). Using the auditory feedback, the subjects were asked to keep the motor unit discharging at a rate of approximately 8 Hz. The reflex responses were recorded using both SEMG and intramuscular EMG while the subject kept the selected motor unit discharging at a constant rate. During the discharge of the selected motor unit, the SEMG levels stayed always below the 5% of the maximal SEMG recorded for that experiment. This low contraction level was similar in our earlier paper in which electrical stimulation was used to study CSP in the same muscle (Kahya et al., 2010). 2.3. Stimulation A laser beam was targeted on the C8 dermatome on the palm of the hand using a CO2-laser stimulator (Neurolas, Electronic Engineering, Florence, Italy). The laser pulses (wavelength = 10.6 lm, intensity = 1.5–15 W, duration = 15 ms) had a beam diameter of 2 mm when irradiating an area of about 3 mm2, with a duration of 15 ms, and a beam diameter of 4 mm when irradiating an area of about 13 mm2. The laser beam was moved slightly to cover the area shown in Fig. 2 (about 10 cm2) after each stimulus to avoid fatigue or sensitization in the nociceptors, where the stimuli were triggered randomly by a computer at intervals of between 5 and 10 s. The cutaneous innervations that supply the C8 myotome, which includes the FDI muscle, are present in the C8 dermatome. The stimulus intensity was increased incrementally up to a level where consistent periods of inhibition were readily observable in the SEMG traces. At least 120 stimuli were used for each trial in the experiments to test 12 motor units. Participants scored the stimuli on an 11-point visual analog scale (VAS) at the end
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2.5. Determination of reflexes The procedure for identifying latency and duration of reflexes was fully described in our earlier paper (Kahya et al., 2010). Briefly, we have determined the prestimulus variations on the CUSUM record to establish the error box (Türker and Powers, 2005; Brinkworth and Türker, 2003). Any post stimulus deflection that was larger than the error box and appeared before the reaction time to the laser pulse stimulus was considered as a significant reflex response to the stimulus. The onset of CSP was marked at the point where the first significant downward deflection occurred in SEMG-CUSUM or PSTH-CUSUM traces. Significant upturn on the other hand is considered to be reflecting the end of the inhibitory period in the probability based analyses. PSF analysis on the other hand uses the same procedure as the PSTH-CUSUM for the onset of the reflex (see the Discussion for the reasons underlying this procedure). The end point of the inhibitory period in the PSF is when the discharge rate of the motor unit returns to the prestimulus levels. Fig. 2. Area for laser stimulation: Laser stimulation was applied to the area of the C8 dermatome on the palmar side (hatched area) and laser handle.
of each trial where 0 indicated no pain and 10 indicated unbearable pain.
2.4. Analyses The SEMG recordings were full-wave rectified and averaged in a 1000 ms window centered on the stimulus. Using the Spike2Ò (CED) program, single motor units were identified and marked using a template matching algorithm. Based on the discharge times of individual motor units, acceptance pulses were created by the Spike2 program and placed into 1-ms bins around the time of the stimulus to construct the PSTH. The PSF is formed by the superimposition of the instantaneous discharge rates of a single motor unit in the temporal neighborhood of the stimuli, where PSF indicates the excitability of the motoneuron membrane (Türker and Cheng, 1994). Thus, the PSFs were constructed using the acceptance pulses obtained from the Spike2 program, and the instantaneous discharge rate values were calculated by taking the multiplicative inverse of the interspike intervals (in seconds). The instantaneous discharge rate values were then placed at the position of the discharge time of the latter spike in an interspike interval. The PSFs were obtained by superimposing the calculated instantaneous discharge rate values around the time of the stimulus using at least 120 stimuli. Cumulative sums (CUSUM; Ellaway, 1978) were obtained using the averaged SEMG to determine the exact timing of the reflex response. CUSUMs were also calculated for PSTH and PSF graphs to detect any subtle but significant changes (Kahya et al., 2010).
2.6. Statistics After confirming the existence of the reflex response using the criteria defined above, we compared the latency and duration values to detect differences between the values calculated with SEMG and PSTH versus PSF methods using Wilcoxon’s signed rank test, where p < 0.05 was considered as significant (Table 1). We also compared the CSP results with our previous results (Kahya et al., 2010) for electrically-induced CSP using Wilcoxon’s signed rank test. 3. Results We obtained 17 SEMG records from 10 subjects and using these 17 records we analyzed results for 12 motor units. This was due to the fact that a minimum of 120 stimuli are needed to obtain reliable single unit analyses and some units disappeared before this stimulus number was reached. According to our experience, large numbers of triggers are needed so that there are sufficient spikes to reliably indicate the changes in the discharge probability and rate around the time of the stimulus (Binboga and Türker, 2012; Yavuz et al., 2014). Significant CSPs were observed in all 17 SEMG recordings. The stimulus intensity that induced CSPs was rated by the participants as between 5 and 6 (mean = 5.46) based on a VAS of 0–10. The mean perception threshold was 65.8 ± 6.5 mJ. The stimulus intensity required to observe CSP was 3–10 times the perception threshold (207.7 ± 12 mJ). The reflex responses were evaluated simultaneously using SEMG, PSTH, and PSF analysis methods, as shown by the example in Fig. 3. The latency of CSP current study was 72 ms for the SEMGCUSUM; and 67 ms for the PSTH-CUSUM. The endpoint values for CSP determined using the probabilistic methods (SEMG and PSTH)
Table 1 Comparison of the latencies and durations of CSP. SEMG recordings and simultaneously recorded single motor units are compared with the values derived from the CUSUMs of SEMG, PSTH, and PSF. SEMG (17 records) Latency of CSP Endpoint of CSP Duration of CSP
72.47 (3.04) 146.59 (7.11) 74.12 (6.16)
PSTH (12 motor units) 67.08 (5.07) 136.08 (6.66) 69.00 (5.79)
PSF (12 motor units) a
67.08 (5.07) 201.67 (7.26)b 134.50 (8.59)
SEMG vs. PSTH p-value*
SEMG vs. PSF p-value*
PSTH vs. PSFa p-value*
0.074 0.113 0.722
0.010 0.010 0.423
– 0.002 0.002
Results represent the mean (ms) ± standard errors shown in brackets. n = motor unit numbers. * Wilcoxon’s signed rank test. a Latency calculations were based on values obtained by the PSTH method. b Endpoint calculations were determined based on the PSF method.
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Fig. 3. Simultaneously recorded reflex responses using two probability-based methods (SEMG and PSTH) and one frequency-based (PSF) analysis methods: Rectified + averaged SEMG and the CUSUM are displayed in the top trace, PSTH and the CUSUM in the middle, and PSF in the bottom trace. From 1 to 4, the vertical lines indicate: (1) position of the stimulus at time zero; (2) latency of the CSP based on the PSTH-CUSUM; (3) end of CSP as indicated by SEMG-CUSUM; and (4) latency of the ‘‘excitatory” period as indicated by SEMG-CUSUM. The endpoint of CSP in PSTHCUSUM and the starting point of CSP in PSF-CUSUM are indicated by downward arrows. The average discharge rate in the PSF-CUSUM record is shown as a solid trace (obtained using moving average of the bin values for 5 ms). The maximum variation in the average discharge rate during the 100-ms prestimulus period is indicated by broken horizontal lines (error box). The y-axis of the PSTH chart shows the number discharges of the motor unit in a given time bin.
were significantly different compared with the values calculated using the frequency-based method (PSF). The duration of CSP was significantly longer when calculated using the PSF method compared with that calculated using the SEMG method (p = 0.002) (Table 1). A typical experiment (original records of single motor units and SEMG) with all of the analyzed variables is illustrated in Fig. 3, where vertical line ‘‘2” indicates the beginning of CSP and vertical line ‘‘3” indicates the termination of CSP determined using SEMGCUSUM. These points were marked using downward vertical arrows in PSTH-CUSUM and PSF average frequency traces. The CSP duration, which was determined as the period between lines 2 and 3 in SEMG-CUSUM, was longer compared with the CSP duration calculated using PSTH-CUSUM, but it was shorter than the CSP duration calculated using PSF. We observed reduced firing frequency values in the PSF graph during inhibition, as shown in Figs. 3 and 4.
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Fig. 4. PSTH, PSTH-CUSUM, and PSF for a single unit from a different subject. The explanations are similar to those given for Fig. 3, where the vertical lines from 1 to 4 indicate the following: (1) position of the stimulus at time zero; (2) latency of CSP based on PSTH-CUSUM; (3) end of CSP as indicated by SEMG-CUSUM; and (4) latency of the ‘‘excitatory” period as indicated by SEMG-CUSUM. The endpoint of CSP in PSTH-CUSUM and the starting point of CSP are indicated by arrows in the average PSF trace. The average discharge rate in the PSF record is shown as a solid trace (obtained using moving average of the bin values for 5 ms). It should be noted that the discharge rate values were lowest in PSF during the ‘‘excitatory” peak in PSTH. The maximum variation in the average discharge rate during the 100-ms prestimulus period is indicated by broken horizontal lines (error box). The y-axis of the PSTH chart shows the number discharges of the motor unit in a given time bin.
In Fig. 4, the differences between SEMG, PSTH, and PSF methods in terms of the CSP duration are further illustrated using a single motor unit from a different subject. Similar points to those described above can also be observed in this figure. Table 2 summarizes our CSP results with those obtained in other studies using both electrical and laser stimulation. 4. Discussion In this study, we obtained several important new results. First, laser stimulation of the hand induced CSP in all of the FDI motor units tested. Second, the duration of the CSP differed according to the analysis program employed. Third, the latency of the CSP elicited using laser stimulation differed from the CSP latency values reported in earlier publications that used electrical stimuli.
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Table 2 CSP in FDI: comparison of current results (bold numbers) and those obtained in other studies (light numbers).
a b
Stimulation site
Latency, ms Mean (SD)
Duration, ms Mean (SD)
Subjects (n)
Authors
Electrical stimulation
Palmar side of the hand (palmar C-8) Digit V (C8 dermatome) Dorsal side of the hand (dorsal C-8)
65 (7) 69.4 (4.34) 58 (2.8) 63.7 (2.8) 70.8 (3.7)
54 (6) 46.7 (6.79) 71.6 (8.1) 74.0 (6.6) 124.5 (11.7)
5 7 6 (18 units) 6 6 (18 units)
Romaniello et al. (2004) Inghilleri et al. (2002) Kahya et al. (2010) (probabilistic-PSTH-CUSUM) Kahya et al. (2010) (probabilistic-SEMG-CUSUM) Kahya et al. (2010) (PSF)
Laser stimulation
Palmar side of the hand (palmar C-8)
90 (7) 67.08 (5.07) 72.47 (3.04) 67.08 (5.07)b
40 (6) 69.00 (5.79) 74.12 (6.16) 134.50 (8.59)
5 10 (12 units) 10 (17 records)a 10 (12 units)
Romaniello et al. (2004) Current results (probabilistic-PSTH-CUSUM) Current results (probabilistic-SEMG-CUSUM) Current results (PSF)
Results were obtained based on 17 SEMG recordings obtained from 10 subjects. Latency calculations were based on values obtained using the PSTH method.
4.1. CSP latency and duration obtained from probability and discharge rate analyses We used probability- and discharge rate-based analyses to investigate the properties of CSP in human FDI muscle induced by painful laser stimuli. In these analyses, the PSF can only provide limited information regarding the onset of an IPSP due to the limited number of discharges during the inhibitory period; therefore, we used the disappearance of the spikes in PSTH-CUSUM to determine the latency of the reflex responses of single motor units for both PSF and PSTH calculations. This approach was explained in detail by Todd et al. (2012) and Ugincˇius et al. (2014). This procedure was also demonstrated to be appropriate for determining IPSP latency in our previous brain slice experiments, where we studied the effect of IPSPs and excitatory postsynaptic potentials (EPSPs) on regularly discharging motor neurons in brain slices (Türker and Powers, 1999). The latency of CSP induced using electrical stimulation in our previous study (Kahya et al., 2010) using the same method was significantly shorter than the latency of CSP in the current study (probabilistic method (SEMG-CUSUM): 63.7 versus 72.5 ms; p < 0.05). In contrast, the termination of CSP is not easily determined via the probability-based classical methods, i.e., SEMG or PSTH. In the classical methods, the termination of the inhibitory period is marked at the time point when a significant activity brings SEMG or bin counts in PSTH back to the prestimulus level or higher following a trough. In the PSF approach, however, the termination of the inhibitory period is determined when the average discharge rate trace returns to the mean prestimulus discharge rate line (Türker and Powers, 1999). This approach was also tested successfully in our regularly discharging rat motor neuron experiments (Türker and Powers, 1999). Fig. 3 and Tables 1 and 2 show that there was a reduction in the discharge rate following the laser stimuli, which had its endpoint 201 ms after the time of the stimulus (time zero). When calculated using the PSF data, the duration of CSP was as long as 134 ms, whereas the CSP duration value calculated using probabilistic methods was 69 ms (also see Romaniello et al., 2004). Depending on the method used, there was a difference of about 65 ms in the calculated CSP durations. The discrepancy between the two calculated values was due to the methodological shortcomings of the probability-based methods when evaluating the duration of inhibitory inputs. We have shown in motoneurons in brain slices that a simple IPSP injected into a regularly discharging motoneuron generates four peaks and troughs in the probability-based analyses such as PSTH. Immediately after the delivery of an IPSP, PSTH indicates a trough followed by a peak in the probability of the spike numbers. Therefore, a peak in the histogram occurs during the rising phase of the injected IPSP (please also refer to Figs. 7 and 8 in Türker and Powers, 1999) and hence places a limit on the duration
of the genuine inhibitory period. SEMG analysis also has similar shortcomings as detailed in Türker and Powers (2003). The PSF analysis on the other hand follows the shape of the IPSP without generating an error in estimating the actual duration of the injected IPSP. Therefore, we are proposing in the current study that the genuine duration of the CSP can be estimated using the PSF method as the PSTH and SEMG methods induce a peak while the IPSP is still continuing and hence underestimate its duration significantly. Furthermore, PSTHs contain secondary peaks and troughs, which are not related directly to the underlying PSP, but instead, they reflect the regular recurrence of spikes (autocorrelogram) following those affected by PSP (Türker and Powers, 1999). PSF analysis is more useful for indicating the total duration and the profile of the underlying PSP. The shape of the underlying PSP can be obtained directly from the PSF records because the discharge rates of the spikes follow the PSPs very closely (Türker and Powers, 1999). The PSF method has been shown to provide a more accurate evaluation of these inputs using known inputs based on regularly discharging motor neurons in rat brain slices (please refer to Figs. 7 and 8 in Türker and Powers, 1999). According to our findings, it is evident that classical knowledge regarding the CSP duration must be revised. A similar result was found for the CSP evoked using electrical stimulation, and its possible implications were discussed by Kahya et al. (2010).
4.2. Laser versus electrical stimulation for inducing the CSP High and low threshold nerve fibers are activated by strong electrical stimuli (Staahl and Drewes, 2004), and thus it would be ideal to use specific stimulation techniques where only the highthreshold fibers are activated to examine the neuronal networks underlying pain pathways responsible for CSP (Plaghki and Mouraux, 2005). Nociceptive laser stimulation of the palm effectively evokes reflex inhibition of the hand muscles, whereas stimulation of the dorsum does not (Romaniello et al., 2004). Laser stimuli activate both myelinated A-delta mechano-heat nociceptors (AMH) and unmyelinated C mechano-heat nociceptors (CMH), but it is not possible to measure the possible contribution of CMH units to the evocation of CSP because the conduction velocity of these unmyelinated fibers is too slow (Romaniello et al., 2004). Type I AMH units require a long-lasting heat stimulus, and the response has a significant delay (in the order of seconds). By contrast, the response of the type II AMH units has a short latency when a short-lasting heat (laser) stimulus is used (Treede et al., 1998). In contrast to type II AMH units, type I AMH units have a higher conduction velocity and lower mechanical thresholds. Thus, due to their corresponding receptor characteristics, it may be expected that AMH I units will respond first to mechanically-
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induced pain, whereas AMH II units will respond first to heatinduced pain (Treede et al., 1995). Laser-induced heat alone is considered to be insufficient to induce an AMH I response. However, the rapid evaporation of water from the outer layers of skin following the laser pulse may act as a mechanical stimulus to activate AMH I units given the low mechanical threshold for these units (Treede et al., 1998). Therefore, the overall effect of heat and the effective mechanical stimuli created by the rapid increase in temperature could be sufficient to excite AMH I units. Electrical stimuli have several advantages because they are easily applicable, controllable, measurable, and safe (Wells et al., 2007). However, they also have disadvantages such as an electrode touching the tissue and tissue impedance attenuating the amplitude of the stimulus current (Geddes and Roeder, 2003). Moreover, the changes in the stimulation threshold depend on the diameter of the fiber, and nerves may be stimulated even before the stimulus reaches the receptors, which might evoke a response that is not unique to the stimulus (Staahl and Drewes, 2004). Thus, an artifact of the stimulus occurs in many records. Moreover, many records contain noise because of the electric fields generated by the stimulus in the area of interest, especially when small amplitude potentials are recorded. By contrast, stimulation using laser beams does not have the disadvantages mentioned above (Plaghki and Mouraux, 2005). However, the effect of the laser stimulus can differ among each subject due to inter-individual variation in factors such as heat absorption, heat loss, and heat diffusion in the skin (Cruccu et al., 2003; Staahl and Drewes, 2004). There is no defined standard for electrical stimulation, but similar results have been obtained for the CSP latency and duration using classical methods for the FDI muscle (Inghilleri et al., 2002; Kofler, 2003; Romaniello et al., 2004) (Table 2). As shown in Table 2, the CSP latency is 63.7 ms using electrical stimulation (Kahya et al., 2010), whereas it is 72.47 ms when laser stimulation is applied (SEMG-CUSUM). During laser stimulation, there is a lag of 8.77ms in CSP compared with electric stimulation (p = 0.039), possibly because electric stimulation of CSP is generated by both A-beta and A-delta fibers (Bromm and Treede, 1991; Treede et al., 1998, 1995), whereas it is induced by stimulation of the A-delta fibers alone during laser stimulation. Therefore, the difference in the CSP delays may be attributable to differences in the conduction time to the spinal cord and/or the number of interneurons in the circuitry. However, part of this difference in the latency may be attributable to differences in the effective stimulus onset time. An electrical stimulus is instantly effective, but the laser stimulus must heat the skin to activate the nociceptors. The exact time required for stimulation of the pain fibers by the laser pulses is difficult to predict, and this must be considered when evaluating the results of this and similar studies using laser stimuli. The electrical and laser experiments were performed at low contraction levels where only low-threshold units were recruited. In both cases, the contraction levels were below 5% of the maximum voluntary contraction and thus similar low-threshold units may be responsible for the inhibition. 4.3. Clinical application The CSP has been utilized in a number of pathological conditions and in a range of experimental settings to assess the conduction of neuronal pathways in patients (Kofler, 2003; Uncini et al., 1991). Furthermore, CSP has been utilized in patients with numerous sensory neuropathies (i.e., Friedreich’s ataxia, abetalipoproteinemia, and Fabry disease). In addition, CSP has been employed to study entrapment neuropathies, including carpal tunnel syndrome (de Tommaso et al., 2009; Kofler et al., 2003; Svilpauskaite et al., 2006). The CSP has been utilized in studies of
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these neurological disorders because changes in CSP may be related to large diameter fiber impairments. For example, delayed CSP latencies have been reported in a small number of patients with neuropathies that involved large fibers (Floeter, 2003; Uncini et al., 1991). However, CSP was always obtained in the absence of fast conducting sensory nerve fibers in the affected nerve; thus, it has been suggested that the A-delta fibers may be responsible for CSP, so studying CSP may provide information about the stability of the A-delta fibers within a nerve. However, as argued in the present study, laser stimulation is the preferred method that can reliably activate A-delta fibers without involving the thicker myelinated fibers. Hence, we suggest that laser stimulation is used when studying nociceptive fiber pathways in basic and clinical research. As shown by the large number of previous studies in this area, researchers have examined the properties of CSP to obtain data about various pathological conditions for both diagnostic purposes and to follow up the progress or regress of these conditions. Standardizing CSP profile using both probabilistic- and frequencybased analysis methods will be crucial for ensuring that this reflex can be used in clinical settings with more confidence. Author contributions KST and MCK designed the experiment. MCK and OS performed the experiments. KST, MCK, and OS analyzed and interpreted the data. KST and MCK drafted the manuscript and all of the authors approved the final version. The experiments were performed at Koç University. Conflict of interest The authors declare that there are no conflict of interests. Acknowledgements We thank all of the subjects who volunteered for this study. Funding: This research was funded by an internal grant from Koç University, Istanbul, Turkey. References Aurora, S.K., Ahmad, B.K., Aurora, T.K., 1998. Silent period abnormalities in carpal tunnel syndrome. Muscle Nerve 21, 1213–1215. Binboga, E., Türker, K.S., 2012. Compound group I excitatory input is differentially distributed to human soleus motoneurons. Clin. Neurophysiol. 123, 2192–2199. Brinkworth, R.S., Türker, K.S., 2003. A method for quantifying reflex responses from intra-muscular and surface electromyogram. J. Neurosci. Methods 122, 179– 193. Bromm, B., Treede, R.D., 1991. Laser-evoked cerebral potentials in the assessment of cutaneous pain sensitivity in normal subjects and patients. Rev. Neurol. (Paris) 147, 625–643. Cruccu, G., Pennisi, E., Truini, A., Iannetti, G.D., Romaniello, A., Le Pera, D., et al., 2003. Unmyelinated trigeminal pathways as assessed by laser stimuli in humans. Brain 126, 2246–2256. de Tommaso, M., Libro, G., Difruscolo, O., Sardaro, M., Serpino, C., Calabrese, R., et al., 2009. Laser evoked potentials in carpal tunnel syndrome. Clin. Neurophysiol. 120, 353–359. Ellaway, P.H., 1978. Cumulative sum technique and its application to the analysis of peristimulus time histograms. Electroencephalogr. Clin. Neurophysiol. 45, 302– 304. Floeter, M.K., 2003. Cutaneous silent periods. Muscle Nerve 28, 391–401. Geddes, L.A., Roeder, R., 2003. Criteria for the selection of materials for implanted electrodes. Ann. Biomed. Eng. 31, 879–890. Inghilleri, M., Conte, A., Frasca, V., Berardelli, A., Manfredi, M., Cruccu, G., 2002. Is the cutaneous silent period an opiate-sensitive nociceptive reflex? Muscle Nerve 25, 695–699. Inghilleri, M., Cruccu, G., Argenta, M., Polidori, L., Manfredi, M., 1997. Silent period in upper limb muscles after noxious cutaneous stimulation in man. Electroencephalogr. Clin. Neurophysiol. 105, 109–115. Kahya, M.C., Yavuz, S.U., Türker, K.S., 2010. Cutaneous silent period in human FDI motor units. Exp. Brain Res. 205, 455–463.
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Mehmet C. Kahya is currently working as an Assistant Professor in the department of Biophysics in the Medical Faculty of Izmir Katip Celebi University where he joined in 2011. Prior to that, he worked as a Medical Doctor in many government organizations. His academic qualification includes a degree from Ege University Medical Faculty in 1989. In 2003, he completed General Surgery residency training and in 2010, he received his biophysics PhD from Ege University. His research area includes cell biophysics, biomechanics, bioelectricity, electrophysiology, electromyography and nociceptive reflex response.
Og˘uz Sebik was born in Izmir, Turkey. He received his bachelor’s degree in Physics in 1998 from Koç University and his master’s degree in psychology in 2000 from University of New Haven. He received his Ph.D. from Ege University in Biophysics in 2012. He is currently employed as a post-doctoral researcher at Koç University, School of Medicine. His current research interests include the investigation of the neuronal pathways involved in motor control, and development of new methodologies to help elucidate the intricacies of motor control in humans.
Professor Kemal S. Türker is a dentist and obtained his Ph.D. degree in Physiology at the Glasgow University, Scotland. He took up a research position in the Medical School of Adelaide University, Australia where he worked from 1983 to 2007. He has then returned to his home country, Turkey as the Marie Curie Chair of the European Union. Kemal has devoted all of his efforts towards understanding the synaptic inputs from receptors to motoneurons that innervate human muscles. Current projects vary from control of human mastication by perioral receptors to modulation of various reflexes during movement.