Muscle spindle discharge modulated by arterial pulsations In previous sections we demonstrated recordings from muscle spindles whose spiking activity was essentially driven by arterial pulsations and spontaneously active afferents in which some spikes apparently became phase-locked to the arterial pulsations. However, for the majority of tonically active muscle spindles – activated by the existing static stretch of the relaxed muscle – a more subtle modulation of discharge activity by the pulse wave was present. In these afferents, a modulation pattern could be discerned in the R-wave triggered average firing rates. This reflected a tendency to increase or decrease their instantaneous discharge rate while individual spikes were not locked to the arterial pulsations. The following analyses were undertaken to identify a proportion of afferents with significant pulse wave modulation, the proportion of total variance explained by the pulse wave, and the common modulatory pattern in the firing of muscle spindle afferents (see Methods). Cardiac rhythmicity detected in the background discharge of muscle spindle afferents. Analyses in this section refer to data obtained from 51 muscle spindle afferents discharging in the absence of active muscle contraction, i.e. in relaxed muscles. The mean discharge rate of the muscle spindles ranged from 3.5 to 23.8 imps/s (8.3 imps/s, median) and the coefficient of variation ranged from 2.0 to 84.7% (8.2%, median). For each muscle spindle afferent we calculated the R-wave triggered average to identify the modulatory effect of the pulse wave on discharge frequency. Figure 4 shows examples of individual traces and averages from 10 muscle spindle afferents (eight innervating primary and two secondary muscle spindle endings). Five of those (A–E) showed increases in discharge rate (positive modulation) at the time of the upbeat of the pulse wave, while five showed an initial decrease (F–J). Note that some afferents had a biphasic modulation profile. Figure 5 shows the equivalent analysis in the frequency domain for the same afferents. The power spectra revealed significant peaks at the cardiac frequency and its harmonics, reflecting periodic modulations of discharge rate at the heart rate frequency. The higher harmonics represent the non-sinusoidal modulation as evidenced in Figure 4. To assess the amount of pulse wave modulation we expressed the variance of the R-wave triggered average as a ratio of the total variance of the discharge rate. The amount of variance explained by arterial pulsations ranged from 0.3 to 61.7% (4.2% median). In 16 out of 51 muscle spindle afferents the variance explained by arterial pulsations was higher that 10%. To statistically assess whether the discharge rate modulation effect was indeed caused by the arterial pulsation - and not by other periodic components in the signal - the level of explained variance was compared to a set of permuted surrogate data (see Methods). Afferents with statistically significant pulse wave modulation In 53% (27/51) of the afferents, pulse wave modulation was significant when evaluated against possible random effects. This effect was statistically significant in 60% (18/30) of the tested Ia afferents and in 43% (9/21) of the group II muscle spindle afferents. For the Ia afferents, the pulse modulation on average accounted for 19.1% of the variance in discharge rate; for group II afferents this was 9.4% (medians 15.3 vs. 7.6%). The largest modulatory effect within the cardiac circle peaked at about 225– 300 ms (median 268 ms) after the R-wave. The mean amplitude of the peak modulation was 10.5% of the mean discharge rate. For the group Ia afferents the peak modulation was 14.3%, while for Modulation of Human Muscle Spindle Discharge group II afferents it was 2.7% in (corresponding medians for group Ia and II afferents were 9.1% and 3.0% respectively). Regardless of the afferent type, the same number of afferents showed a positive or negative initial peak (14 vs. 13 afferents respectively). In 70% (19/27) of the afferents the modulation was biphasic and the initial peak was followed by a second peak of opposite polarity. The median amplitude of the second peak was 68% of the initial peak. The relative size of the peak modulation correlated inversely with the mean discharge rate (rs = 20.54, p,0.05), while the absolute size of modulation expressed as imp/s was not influenced by discharge rate (rs = 20.13, p.0.05); this indicates that pulse wave modulation was largely additive. The relative size of the peak modulation showed a positive correlation with the coefficient of variation of discharge rate (rs = 0.80, p,0.05; Spearman’s rank correlation test). There was a weak relationship between cardiovascular parameters and the size of the modulatory effect across different subjects and afferents: the relative size of the peak modulation showed a weak correlation with mean blood pressure (rs = 0.46, p,0.05), but not with pulse pressure (rs = 0.15, p.0.05). The amount of variance explained by arterial pulsations was not correlated either with mean discharge rate (rs = 20.10, p.0.05) or coefficient of variance (rs = 0.33, p.0.05). Effects at the level of muscle spindle afferent population assessed by PCA To identify common features of the modulatory effect we used principal component analysis (PCA) to compare discharge rate modulation patterns across afferents. Most of the modulatory pattern features were explained by the first principal component (p,0.05): 48% of modulatory effect was accounted by the same pattern sharing common features between different afferents (Fig. 6A). These analyses revealed a tri-phasic modulation pattern with three stable peaks reflecting increases and decreases in discharge rate. The distribution of the corresponding eigenvector coefficients confirmed that the arterial pulse wave had either an excitatory or an inhibitory effect on different afferents, as reflected by a positive and negative coefficient, respectively (Fig. 6B). Presumably, the orientation of the muscle spindle ending with respect to a nearby blood vessel may result in loading (stretch), thereby increasing its firing rate, or unloading, and hence decreasing its discharge rate. The second principal component explained 21% of the variance (not shown) and was a modulation of the first principal component that captured the difference in latency of the modulation effect across afferents. PCA of the power spectra further demonstrated the effect of arterial pulsations on muscle spindle discharge at the population level. The first principal component (p,0.05) explained 43% of the variance and revealed stable periodic modulation at the heart rate frequency and its harmonics (2f and 3f). The second principal component (explained variance 12%) was again a modulation of the first principal component, reflecting differences between afferents (not shown). For both time- and frequency-domain analyses, the afferents showing significant pulse wave modulation (determined from data resampling using circular permutation, as described in Methods) had larger eigenvector coefficients, as illustrated by the black histograms in Fig. 6BD. Effect of physiological and cardiovascular challenges on the nature of pulse wave modulation of muscle spindles. An important question we intended to address is how much the strength of the pulse wave modulatory effect changes during physiological challenges that cause specific changes in cardiovascular parameters, sympathetic outflow or gamma motor neuron activity. In the following section our focus is PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35091