sympathetic fibers but is expressed in nociceptive afferents. In addition, DRG neurons express higher levels of Kv1.1, Kv 1.8, and Kv 3.1 than sympathetic neurons (Munns and Koltzenburg, personal communication). The depolarizing afterpotential in sympathetic neurons may also be due to activation of Cl- channels via voltage-dependent Ca++ channels [29]. Relative Speeding Develops in Afferent Fibers During the course of the repeated twin-pulse stimulation, the twin pulse speeding became more pronounced in the efferent fibers. The afferent fibers, which initially showed twin-pulse slowing, developed a relative twin-pulse speeding (e.g., Fig. S2). Sympathetic efferents exhibited absolute speeding of the second action potential when pulses of twin stimuli were separated by intervals of 20–100 ms. This speeding increased with ongoing stimulation but reached a plateau after about 20 twin pulses. In contrast, mechanosensitive afferents never showed absolute speeding regardless of the stimulus interval used; however, relative speeding developed during ongoing stimulation at stimulus intervals of 20–100 ms. Thus, at these stimulus intervals, the axonal hypoexcitability which is apparent as conduction slowing of the trailing action potential at the beginning of the stimulation decreases and is replaced by axonal hyperexcitability that becomes evident as relative speeding. At the end of the stimulation with 60 twin pulses, slowing of the leading action potential was significantly larger in afferent than in sympathetic efferent fibers. In both fiber classes, the latency difference between the leading and trailing action potential correlated negatively with the increase in latency of the last leading action potential. Furthermore, the time course for the slowing of the leading action potential was similar to the time course for the speeding of the trailing action potential for both the sympathetics and the afferents (Fig. S4B,C). Taken together these findings suggest that related mechanism(s) may underlie the phenomena of activity-dependent slowing and relative ‘speeding’. As hypothesized earlier [9,11], activity dependent hyperpolar- ization increases sodium influx and reduces potassium outflux during an action potential thereby creating a net depolarizing charge on the axonal membrane and resulting in a depolarizing afterpotential. Our observation that slowing and relative speeding have a similar time course is in agreement with this hypothesis. The exponential recovery from relative speeding for both the afferents and the sympathetic efferents is consistent with a discharge through the membrane RC circuit. The time constants for recovery for the sympathetics (137 ms) and the afferents (89 ms) are comparable to those reported previously [9,11]. Conduction Criterion to Distinguish Afferents from Sympathetic Efferents A fiber that is insensitive to mechanical stimuli applied to its cutaneous terminals can be either a sympathetic efferent or a mechanically-insensitive afferent. Positive classification of an afferent requires the identification of the adequate stimulus which may be one of many noxious chemicals (e.g., mustard oil, capsaicin, histamine) or thermal stimuli (cold, heat). To avoid applying multiple noxious agents to the skin, it would be useful to have an independent criterion for identifying sympathetics. Our finding that only sympathetic efferents show absolute speeding of conduction to a twin pulse (ISI = 50 ms) provides such a criterion. Supporting Information Figure S1 Voltage-latency curve for an afferent obtained by electrical stimulation at the receptive field using the well electrode. Conduction in Small Fibers Before starting with the electrical stimulation protocols at the cutaneous terminals, electrical stimuli of constant duration (1 ms) but of increasing intensity were applied every 4 s. Threshold for electrical activation and the latency at this stimulus intensity were measured. Intensity was increased until a step decrease in conduction latency was observed. The stimulus intensity necessary to produce this step and the resulting new latency were noted. Intensity was increased to the upper limit of the Grass constant- voltage stimulator (150 V) or the Digitimer Constant Current stimulator (100 mA). Stimulus intensities and conduction latencies were used to generate voltage-latency curves similar to the example shown in this figure. At this stimulation site, the electrical threshold for activation was 60 V, and the resulting conduction latency was about 125 ms. Up to an intensity of 85 V, the conduction latency was stable, but at an intensity above 85 V, the latency stepped down to about 123 ms. Another latency step was observed with stimulus intensities above 99 V at which the latency decreased to about 115 ms. No additional decrease in conduction latency was observed up to an intensity of 150 V. These different latency levels correspond to discrete action potential initiation sites within the cutaneous arbor of the afferent. The purpose of these voltage-latency curves was to identify a wide stimulus intensity window over which the latency of the unit under study was stable to insure that the AP initiation site was fixed. For the subsequent electrical test protocols, stimulus intensity was usually set half way between the upper and lower limits of such a stable window. For this particular fiber, the stimulator was set at 125 V. Found at: doi:10.1371/journal.pone.0009076.s001 (0.01 MB PDF) Figure S2 Presumed sympathetic fiber shows absolute and relative speeding to 60 twin pulses. A recording from a C fiber is schematically summarized in Fig. 2A. For this presumed sympathetic fiber, transcutaneous electrical stimulation consisted of twin pulses delivered every 2 s with a stimulus interval between pulses of a twin stimulus of 50 ms. In response to twin stimulus #1, the first action potential (AP) arrived at the recording electrode 123 ms after the delivery of the first pulse at the cutaneous receptive field (see left vertical dashed line). Since the stimulus interval was 50 ms, the 2nd AP was expected to arrive at the recording electrode at least 173 ms after the first pulse. In other words, the 2nd AP was expected to arrive at the recording electrode about 50 ms following the 1st AP (indicated by the right vertical dashed line in Fig. 2A). However, the 2nd AP arrived at the recording electrode 171.8 ms after the first pulse, correspond- ing to a latency of 121.8 ms from the second pulse. Thus, the conduction of the 2nd AP was 1.2 ms faster than the conduction of the ‘‘na¨ıve’’ AP (defined here as the first AP in the stimulus train). This phenomenon represents absolute ‘‘speeding’’ of conduction by 1% (marked by ‘‘A’’ in the trace for twin stimulus #1). For the second twin stimulus, the latency of the 1st AP (i.e., AP #3 in train) increased, as did the latency of the 2nd AP (AP #4). However, the latency of AP#4 was still smaller than the latency of the naive AP, indicating that absolute speeding still occurred. The relative latency of the 1st AP (S in figure) and the 2nd AP continued to increase during the course of the stimulation. At the end of the stimulus train (i.e., twin stim #60), the latency of the 1st AP (i.e., AP# 119) had increased to 129 ms (corresponding to a slowing of 4.9% relative to the na¨ıve AP), whereas the latency of the 2nd AP (i.e., AP#120) had only increased to 124 ms. The latency of the 2nd AP was still shorter than the latency of the 1st AP, but it was now longer than the latency of the naive AP. Thus, the 2nd AP no longer showed absolute speeding, but it did show relative speeding with respect to the 1st AP (marked by ‘‘R’’ in the trace for twin pulse #60). The latencies of the two APs are plotted PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9076