as a function of twin stimulus number in B. The latency of the naive AP (i.e., AP #1) is indicated by the horizontal line in the graph. The latency of the 2nd AP was shorter than the latency of the naive AP up to twin stimulus #14, indicating the presence of absolute speeding (see arrow labeled ‘‘A’’). Throughout the stimulus train, the latency of the 2nd AP was smaller than the latency of the 1st AP, indicating the presence of relative speeding (see arrow marked ‘‘R’’). The relative latency of the 1st AP (S) increases throughout the protocol indicating activity-dependent slowing. In C, the difference between the latency of the 2nd AP and the naive AP is plotted as a function of the twin stimulus number (filled symbols). For the first 14 pairs of twin pulses, this difference was negative indicating absolute speeding. For the remaining twin stimuli, this difference became positive as the latency of the 2nd AP in a given twin stimulus became larger than the latency of the naive AP. C also shows the latency difference between the 2nd AP and the 1st AP for every twin stimulus (i.e., the latency difference between twin APs, open symbols). Since the 2nd AP of a given twin stimulus always had a smaller latency than the preceding 1st AP of the same twin stimulus, the latency difference between twin APs was always negative, indicating the presence of relative speeding throughout the stimulation. As can be seen, this difference became more negative during the first 25 pairs of twin stimuli after which it reached a plateau. As shown in B, this plateau is due to the fact that the conduction latencies of both action potentials had reached a plateau by the 25th pair of twin stimuli, and that they increased only minimally throughout the rest of the train. To analyze the latency data across different fibers, the latency difference between twin APs of a given twin stimulus was normalized by dividing by the latency to the naive AP of that stimulus train and is referred to as the ‘‘twin pulse difference.’’ In D, the twin pulse difference data for this fiber are plotted as a function of stimulus intervals (10, 20, 50, 100, 200 and 500 ms). Each curve corresponds to data collected at a given twin stimulus number during the different trains (i.e., twin stimulus number 1, 10, 20, 40, 60). In this fiber, the stimulus intervals of 10 and 500 ms resulted only in positive latency differences, indicating that the 2nd AP was always conducted slower than the 1st AP of the twin stimulus. Moreover, the positive difference did not change markedly from the first to the 60th twin stimulus. In contrast, the latency differences between twin APs collected at the stimulus intervals of 20–200 ms were all negative. Specifically, this difference was negative with the first twin stimulus, i.e., absolute speeding was observed at these intra twin pulse intervals. Furthermore, the latency difference between twin APs became more negative for the first 20 twin stimuli, after which the difference reached a plateau and no further increase was observed. C-fiber mechanosensitive nociceptive afferent shows only relative speeding (E-H). The right hand column shows a recording in a typical C-mechanosensitive afferent (C-MSA). The interval between the twin pulses was 50 ms, and twin stimuli were applied every 2 s. As shown in E, the second AP in response to twin stimulus #1 was conducted slower than the naive AP. With ongoing stimulation, the latencies of both APs increased, i.e., the conduction for both action potentials slowed. However, at the end of the stimulus train, the second AP (AP # 120) occurred less than 50 ms after the first AP (AP #119), indicating relative speeding of the second AP. When the latencies for the first and second action potential are plotted separately (see Fig. 2F), the development of relative speeding becomes more evident. For the first 11 twin stimuli, the latency of the second AP was greater than the latency of the naive AP which is marked by the horizontal line. In addition, the latency of the second AP was larger than the latency of its preceding AP. At twin stimulus #12 the latencies from both Conduction in Small Fibers APs were almost identical. After the 12th twin stimulus, the latency of the first AP became larger than the corresponding second AP, i.e., relative speeding (R) developed. At the end of the stimulus train, the latency of the first AP had increased by a total of 20 ms to 186 ms, whereas the latency of the second AP had only increased by 9 ms. Fig. 2G shows the latency differences between the second AP of a twin stimulus and the naive AP (filled symbols). Since the second AP was always conducted slower than the naive AP, this difference was always positive. This is in contrast to the latency difference between the second and its preceding AP of a given twin stimulus which is represented by the open symbols in Fig. 2G. In the beginning of the stimulus train, this difference was positive, but with ongoing stimulation, it became smaller. With twin stimulus #12, this difference became negative, again illustrating the development of relative speeding. Relative speeding increased over the stimulus train and at the end of the stimulation the second AP was conducted 14 ms faster than the preceding AP. Fig. 2H summarizes the normalized data that were collected for the different intra twin pulse intervals used. As can be seen, the latency difference between the twin APs of the twin stimulus #1 was always positive regardless of the stimulus interval, i.e., slowing of the second AP was always observed. Between twin stimuli 10 and 20 the difference became negative indicating the development of relative speeding. For all stimulus intervals between 20–200 ms, relative speeding increased with the number of twin stimuli applied. No major changes in conduction latency were observed when the stimulus interval was set at 500 ms. Found at: doi:10.1371/journal.pone.0009076.s002 (0.09 MB PDF) Figure S3 Twin pulse data from mechanically-sensitive afferents and presumed sympathetic efferents. Fifteen presumed sympa- thetic fibers and 10 mechanically-sensitive afferents were studied with 2 or more different stimulus intervals using the repeated twin pulse paradigm. The twin pulse difference for the first twin pulse (A and B) and the last twin pulse (C and D) are plotted for these presumed sympathetic fibers (A and C) and mechanically-sensitive afferents (B and D). Each line corresponds to a different fiber. Found at: doi:10.1371/journal.pone.0009076.s003 (0.07 MB PDF) Figure S4 Time constants. A. Recovery from twin pulse speeding. The average twin pulse difference for the last twin pulse is plotted as a function of stimulus interval for the mechanically-sensitive afferents (red triangles) and the presumed sympathetic fibers (blue circles). Exponential fits to the data over the interval from 50 to 500 ms are indicated by the dashed curves. The recovery time constant for the mechanically-sensitive afferents (89 ms) was shorter than for the presumed sympathetic fibers (137 ms). B. Activity- dependent slowing during the repeated twin pulse stimulation. The relative increase in latency of the first action potential in the twin pulse is plotted as a function of time during the twin pulse paradigm that lasted for 120 s for the mechanically-sensitive afferents (red triangles) and the presumed sympathetic fibers (circles). Exponential fits to the data are indicated by the dashed curves. Most of the activity-dependent slowing of the sympathetic fibers occurred in the first 20 pulses (time constant = 27 s). The activity-dependent slowing in the mechanically-sensitive afferents continued to increase throughout the paradigm (time constant = 64 s). The activity- dependent slowing of the first action potential was not dependent on the ISI of the twin pulse. C. Twin-pulse speeding during the repeated twin-pulse paradigm. The twin-pulse difference is plotted as a function of time during the twin-pulse paradigm. Average data for the 50 ms stimulus interval are used. For the presumed sympathetic fibers (blue circles), the twin-pulse difference starts PLoS ONE | www.plosone.org 11 February 2010 | Volume 5 | Issue 2 | e9076