Figure 5. Examples of spectral analyses for the same ten afferents illustrated in figure 4 A–J, respectively. The gray shaded area shows the distribution of spectral power represented as a percentage of total power. Mean heart rate (HR) is indicated by the left dashed vertical line and numeric value. The right dashed vertical line indicates the frequency twice that of the heart rate. The thin black line indicates the 95% confidence interval, determined using the surrogate power spectrum distribution. doi:10.1371/journal.pone.0035091.g005 on possible population effects and converging inputs from muscle spindles. Accordingly, we primarily report mean values while acknowledging that distributions might be skewed (see Methods). Inspiratory capacity apnoea This physiological manoeuvre - a maximal inspiratory breath- hold - is known to cause a sustained increase in muscle sympathetic nerve activity. The manoeuvre typically caused an Modulation of Human Muscle Spindle Discharge initial fall in systolic blood pressure typically for up to 25 mmHg and a sustained fall in pulse pressure by 9 mmHg in average. Muscle spindle discharge parameters were quantified during 30 s intervals measured in 22 spindle afferents during rest and apnoea. Ongoing discharge rate and discharge variability of muscle spindles were not influenced by the manoeuvre. This was true for nine primary and thirteen secondary afferents analysed separately (p.0.05; Wilcoxon test). Moreover, the overall size of discharge rate variability explained by arterial pulsations did not change (7.0 vs. 7.4% during control condition and apnoea respectively; p.0.05 Wilcoxon test), nor did modulation strength of single afferents (p.0.05; Wilcoxon test). Indeed, the average discharge modulation was similar during the apnoea and control period for both positively (Fig. 7A) and negatively modulated afferents (Fig. 7B). There were no differences when group Ia and II afferents were analysed separately. Also, the strength of peak modulation was not influenced by the manoeuvre (6.0% vs. 6.1%; p.0.05; Wilcoxon test). The same conclusion was supported by analyses conducted on only those afferents that were significantly modulated (9/22) were selected: neither the amount of explained variance (14.1 vs. 15.1%) nor the size of peak modulation (8.6 vs. 8.3%, for control and apnoea respectively) were influenced by the apnoea (p.0.05; Wilcoxon test). Acute pain We investigated whether pulse wave modulation was affected by acute muscle or skin pain. In 12 afferents investigated with muscle pain eight afferents showed significant modulation by arterial pulsations. Thirteen afferents were tested with skin pain, from which seven afferents were significantly modulated. On average, there was a small decrease in discharge rate during muscle pain (9.9 vs. 9.4 imp/s) and there was no effect of skin pain on the mean discharge rate of the muscle spindles (9.6 vs. 9.5 imp/s). The amount of variance explained by arterial pulsations was not affected by muscle pain (16.0% vs. 15.6% for control and pain respectively; p.0.05, n = 12; Wilcoxon). With skin pain the amount of variance explained by arterial pulsations on average decreased from 8.3 to 5.3%. A decrease was observed for eight out of 13 afferents but the overall effect was not significant at the population level (p.0.05; Wilcoxon; Fig. 7EF). However, from seven significantly modulated afferents the amount of explained variance decreased in six afferents and this effect was significant (p,0.05, n = 7; Wilcoxon). No changes were detected in the amount of overall discharge variability between background and muscle- or skin-pain conditions (p.0.05; Wilcoxon). Changes in the pulse wave modulatory effect observed during the pain were not correlated with changes in heart rate (p.0.05) or changes in pulse pressure (p.0.05, n = 25; Pearson correlation). In sum, these analyses indicate that no or only very weak effects from muscle or skin pain could be detected. Active contraction. For seventeen muscle spindle afferents (14 primary and 3 secondary) cardiac rhythmicity was investigated during moderate strength voluntary muscle contraction. Twelve afferents (71%) showed significant modulation of discharge rate by arterial pulsations. All afferents were silent in relaxed muscles, but became engaged during active contraction most likely due to co- activation of gamma (fusimotor) neurones. Mean discharge rate during the active muscle contraction was comparable to the mean discharge rate observed in spontaneously active muscle spindles (10.2 vs. 9.6 imp/s, respectively); however, the coefficient of variance was higher during active contraction (21.7 vs. 10.8%). Due to this higher overall discharge variability the size of the explained variance was relatively small (Fig. 7GH). That is, only 2.4% of total variance was explained by arterial pulsations. In PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35091