Figure 7. Comparison of R-wave triggered discharge rate averages between different physiological conditions. Modulation of discharge rate is normalized to the variance in discharge rate over the whole recording and plotted as a function of time, measured from the R- wave of ECG signal. Afferents are grouped according to whether the initial modulatory peak in response to the upbeat of the pulse wave is positive (graphs in left column) or negative (graphs in right column), as revealed by the sign of the eigenvector coefficient (Fig. 6B). A & B, changes in discharge rate modulation during inspiratory-capacity apnoea. C & D, changes in discharge rate modulation during muscle pain. E & F, changes in discharge rate modulation during skin pain. G & H, discharge rate modulation during active voluntary contraction. doi:10.1371/journal.pone.0035091.g007 Modulation of Human Muscle Spindle Discharge neurons. That is, functionally independent afferents may show synchrony and phase locking to a common periodic source of input, such as cardiac rhythmicity. Of the spontaneously active afferents that showed significant pulse wave modulation (27 of 51 afferents), on average the amplitude of peak modulation was above 10% of discharge rate, explaining 15% of the total variance in discharge rate. Hence, even for modulated muscle spindles the pulse wave is a significant factor that determines their output behaviour. Modulation of discharge rate during apnoea and pain One of the central questions in the current study was to evaluate the extent to which changes in hemodynamic parameters and sympathetic activity influence the strength of pulse wave modulation. These investigations were aimed at responding to speculations that changes in cardiovascular parameters may have an impact on the noise level in the discharge of muscle spindles and thus influence their proprioceptive function and even lead to clinically significant consequences. First, we tested effects caused by an inspiratory-capacity apnoea [19]. In contrast to our expectations, the apnoea did not significantly alter the depth of pulse wave modulation. This finding may seem surprising as the apnoea is a very potent stimulus which causes a persistent fall in pulse pressure, a sustained increase in muscle sympathetic nerve activity and a decrease in muscle blood flow. However, this finding agrees somewhat with previous observations of McKeon and Burke [4], who could not fully eliminate cardiac rhytmicity in muscle spindle discharge even when occluding arteries by cuff inflation. One of the explanations may be related to the fact that conductance of the vessel increases in proportion to the fourth power of its diameter and there might be little change in geometry between the muscle spindle organ and the neighbouring blood vessels. Also, local biomechanical and physiological mechanisms are likely to contribute to dampening the effect of changes in systemic blood pressure. On the other hand, those observations also suggest that even very strong increases in muscle sympathetic nerve activity have no direct effect on the sensitivity of muscle spindles. This confirms previous findings in humans [12], but differs from observations obtained in anaesthetised cats [13,14]. Our findings indicate that pulse wave modulation of muscle spindle discharge is robust and relatively insensitive to spontaneous fluctuations in blood pressure. Apparently, they do not show any direct response to activation of sympathetic efferents, for which there is some evidence of direct innervation of muscle spindles [33,34]. Any major changes in muscle spindle discharge modulation by arterial pulsations will thus most likely reflect changes in activation of gamma motor neurones. This is an important conclusion, long sought after by researchers investigat- ing various effects of autonomic and emotional systems on proprioceptive function; pain is one such example, given its widespread effects. Furthermore, from a methodological point of view this suggests that cardiac modulation of muscle spindles could be used as a reliable indicator of muscle spindle sensitivity to dynamic stimuli. The advantage of such a weak but reliable natural dynamic stimulus is its robustness, as it does not cause any mechanical changes in muscle properties – unlike the repetitive muscle stretch typically employed to assess dynamic stretch sensitivity. We exploited this feature to detect subtle changes in dynamic sensitivity of muscle spindles during acute muscle or skin pain. Animal studies have shown that noxious inputs onto gamma motor neurones can cause an increase in the activity of muscle spindles. It has been proposed that this may cause a fusimotor-driven increase in muscle stiffness and thus may underlie the development of PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e35091