Figure 6. Mechanically-sensitive afferents and presumed sympathetic efferents exhibit different twin pulse speeding and activity-dependent slowing during the repeated twin- pulse paradigm. A) Presumed sympathetic efferents. The twin- pulse latency difference is plotted versus stimulus interval for the 1st, 5th, 10th, 20th, 40th and 60th twin stimulus. Twin pulse speeding is observed for ISIs between 20 and 100 ms. The amount of twin pulse speeding increases during the repeated paradigm (n = 13–20, except at the 1st twin pulse at 50 ms where n = 32). B) Mechanically-sensitive afferents. For the first twin pulse, twin pulse slowing is observed over all ISIs. After 20 twin pulses, speeding develops over a range of ISIs from 20 to 100 ms. (n = 7–12, except at the 1st twin pulse at 50 ms where n = 46). C) The plot of final twin pulse difference versus final slowing reveals two non-overlapping clusters corresponding to the presumed sympathetic efferents and the afferents. Presumed sympathetic efferents and afferents exhibited a similar range of twin-pulse speeding by the final (60th) twin pulse of the repeated Conduction in Small Fibers twin pulse protocol. The final activity-dependent slowing (i.e., increase in latency of the first AP in the last twin-pulse relative to the latency of the first AP in the train) was greater in the sympathetics than the afferents. One mechanically-insensitive fiber (green circle) not tested with chemicals behaved like an afferent. Only data from twin pulses separated by 50 ms are included. Symbol coding same as in Fig. 3C. D) Correlation of the final slowing and conduction velocity. The final slowing tends to decrease as the conduction velocity increases for both classes of fibers. Mechanically-sensitive afferents and presumed sympathetic efferents form distinct clusters. Note that the green circle is again within the afferent cluster and presumably a mechanically- insensitive afferent. All data in this figure were obtained in centripetal recordings. doi:10.1371/journal.pone.0009076.g006 interval used, no speeding was observed for the first twin pulse. 2) At the smallest stimulus interval (i.e.,10 ms), only slowing was observed. With increasing number of twin stimuli, the magnitude of the twin pulse difference became smaller but remained positive throughout the stimulus train. 3) For intervals between 20 and 100 ms, slowing was present for the first 20 twin pulses. After 20 twin pulses, twin pulse speeding developed and the magnitude of the speeding continued to increase for the remainder of the twin pulses (See also Fig. S3C). 4) The largest speeding occurred at the 50 ms stimulus interval; the maximum value for the speeding in MSAs (3.0 + 1.0%) occurred at the end of the train and did not differ from the speeding in presumed sympathetic fibers (p.0.05). Final Twin-Pulse Speeding Is Correlated with Final Activity-Dependent Slowing Previous microneurography studies in humans [21] suggested that relative speeding in C- fiber nociceptors is correlated with the amount of slowing of the first action potential. To investigate if such a relationship is also seen in C- fibers of non-human primate, the twin pulse difference of final twin stimulus (twin pulse #60) was plotted against the total amount of slowing for the first action potential of the final twin pulse (i.e., the latency difference between action potential #119 and action potential #1). Only data from stimulation with an ISI of 50 ms were used. Fig. 6C summarizes these data for the different classes of fibers that were investigated in this study. In both classes of C- fibers, the amount of twin-pulse speeding at the end of the stimulus train was correlated with the amount of total slowing at the end of the stimulation. The average final slowing for the mechanically-sensitive afferents (9.9 + 0.9%) was significantly greater than for the presumed sympathetic fibers (4.3 + 0.4%, p,0.001), but, as noted above, the average amount of final twin pulse speeding did not differ. Notably, the presumed efferent fibers and afferent fibers formed two non-overlapping clusters. The latency difference after the final twin pulse latency was correlated to the difference after the first twin pulse (Fig. S5). Thus, sympathetic fibers that exhibited more initial twin pulse speeding developed more twin pulse speeding during the repeated stimulation than fibers with little initial twin pulse speeding. A plot of final slowing versus conduction velocity (Fig. 6D) reveals two partially overlapping clusters. The fiber marked by the green circle is in the middle of the afferent cluster in figures 6C, 6D, and 3C, and thus likely is a mechanically-insensitive afferent. Discussion In the peripheral nerve, unmyelinated sympathetic efferent fibers and unmyelinated primary afferent fibers are known to exhibit different compositions of voltage gated K+ and Na+ ion channels. This suggests that their conduction properties may differ. Using PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9076