prevailing direction of differences within population. In this test each afferent was represented by pair of values representing two different conditions – control versus apnoea or pain. The Pearson coefficient of correlation (r) was used as measure of correlation. As a nonparametric alternative and measure of correlation, we used the Spearman rank correlation coefficient (rs). In all tests, the level of probability selected as significant was p,0.05. Unless otherwise indicated, population estimates are presented as mean values. Results Afferent sample Unitary recordings were made from a total of 75 muscle spindle afferents in 40 subjects. Discharge activity in 58 muscle spindle afferents showing spontaneous activity was recorded from relaxed leg muscles while voluntary muscle contraction was required to activate the remaining 17 afferents. Based on behavioural criteria 50 afferents were classified as supplying primary endings (group Ia afferents) and 25 as supplying secondary endings (group II afferents). Muscle spindle firing driven by arterial pulsations In total, we encountered seven muscle spindles discharging at a relatively low discharge rate (#3 imp/s), apparently driven by arterial pulsations, e.g., showing no other spontaneous activity than spikes generated with a clear cardiac rhythmicity. Four muscle spindles fired one spike per cardiac cycle, corresponding approximately to the ascending section of the pulse wave (anacrotic limb or upbeat), which occurs about 250 ms after the peak of the R-wave of the ECG (Fig. 1). R-wave triggered post- stimulus time histograms revealed that every spike generated in these afferents was locked to the R-wave and thus exclusively driven by the arterial pulse, as shown for the three units in Fig. 2A–C. We also encountered 3 muscle spindles that fired two or three spikes per cardiac circle. As shown in Figure 2D–F, the second spike usually closely followed the first. Two of those muscle spindles (Fig. 2E–F) showed a second cluster centred at the latency corresponding to the dicrotic notch of the pulse wave (cf. Fig. 1). Modulation of Human Muscle Spindle Discharge Phase locking of ongoing spindle discharge For the majority of spontaneously active afferents the ongoing discharge was primarily evoked by muscle stretch properties other than the pulse wave. However, the effects of cardiac rhythmicity could be discerned also in those afferents. The most pronounced effect was phase locking of individual spikes. In the literature such kind of behaviour is referred to as ‘‘resetting,’’ and is described as an afferent that ‘‘discharged or failed to discharge at a fixed interval after the pulse’’ [4]. Thus, such behaviour was predicted and searched for [4], but has not been previously found. No formal criteria have been developed to distinguish such afferents, therefore we relied on visual inspection of spike alignment in rasterplots and peak-and-trough patterns in density histograms. Such behavior is rarily seen in muscle spindle afferents, however three afferents clearly stood out. For example, the primary spindle afferent shown in Fig. 3A discharged spontaneously at a low rate, around 10 imp/s. At 300 ms after the R-wave, corresponding to the ascending portion of the pulse wave, its instantaneous discharge rate exceeded 30 imp/s. After the peak there was a silent period of about 200 ms when the discharge virtually ceased. This is a very typical characteristic of dynamically sensitive group Ia muscle spindle afferents – they usually exhibit a silent period following an excitatory stimulus [31,32]. Spiking activity resumed after 650 ms; this latency was constant across trials. In this subject, the R-R interval was 0.95 s and, due to the heart rate variability, the raster plot showed substantial jitter at the onset of the next systolic wave. Thus, in this case, phase locking of spiking activity resulted from post-stimulus depression. In another Ia afferent phase locking was achieved through a different mechanism. As shown in the example illustrated in Fig. 3B, phase locking occurred because of the excitatory effect of the upstroke of the pulse wave (Fig. 3B). Three spikes generated at the time of the upstroke of the pulse wave showed slightly increased discharge rate and tight phase locking regardless of the timing of the preceding spike. The first inter-spike interval following the triplet was slightly prolonged, after which regular spiking activity resumed. R-triggered raster plots showed that these spikes were well aligned, although there was more jitter than during the pulse wave period. Figure 1. Example of muscle spindle discharge locked to the arterial pulsations. This afferent responded with one single spike at the early part of the upbeat of pulse wave and about ,250 ms following R-wave in ECG signal. Note also muscle sympathetic burst activity in nerve signal. doi:10.1371/journal.pone.0035091.g001 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35091