(10, 20, 50, 100, 200 or 500 ms). Trains of twin stimuli were separated by a stimulus free interval of at least 2 minutes. The stimulation protocol was started with twin pulses separated by 50 ms. The order of the remaining trains was randomized. A schematic of the stimulus protocol is presented in Fig. 1C. Data Collection and Analysis A personal computer with a computer-based data acquisition board and customized data acquisition and analysis software (DAPSYS; Brian Turnquist; see www.dapsys.net) was used to display, record, and store action potential activity. On- and off-line action potential discrimination could be performed using DAPSYS. The electrical stimulators were triggered by signals from the DAPSYS software. Recorded action potentials and trigger pulses were time stamped in DAPSYS to allow for direct correlation between the two. Data are presented as mean + S.E.M. Data were normalized to combine latency changes across fibers: a percent change in latency was computed by dividing the latency difference by the latency of the first action potential in the stimulus protocol. Student’s t-tests were used to compare data (except where noted otherwise). Sigma Plot was used to determine the linear and exponential regression lines and to determine significance of linear correlations. Results A total of 48 C-fibers in the superficial peroneal nerve that were activated by electrical stimulation of the sciatic nerve were positively identified as originating from the dorsal root using collision techniques; these afferent fibers had identical mean conduction velocities when measured from the dorsal root (1.04 + 0.03 m/s) or from the sciatic nerve(1.04 + 0.03 m/s) electrode. Similarly, 24 C- fibers in the superficial peroneal nerve were positively identified as originating from the sympathetic chain; these sympathetic efferent fibers had similar mean conduction velocities when measured from the sympathetic chain (1.02 + 0.09 m/s) and from the sciatic nerve (0.96 + 0.05 m/s) electrode. A total of 82 C-fibers was investigated in the centripetal experiments in which the cutaneous terminal was electrically stimulated. Of these, 46 were responsive to mechanical stimuli (von Frey threshold = 2.4 + 0.2 bar) and had a mean conduction velocity of 0.95 + 0.02 m/s. The remaining 36 fibers were insensitive to mechanical stimuli and had a mean conduction velocity of 0.87 + 0.04 m/s. Sympathetic Efferent Fibers Exhibit Supra-Normal Conduction In response to two electrical pulses (inter pulse interval 50 to 70 ms) delivered at the sciatic nerve electrode, most of the sympathetic efferent fibers exhibited a speeding of conduction; the second action potential was conducted at a faster conduction velocity than the first action potential. For the example shown in Fig. 2A, the interstimulus interval was 60 ms. The action potential at a latency of 148.6 ms (waveform # 2) was positively identified as belonging to a sympathetic efferent based on collision experiments (see methods). The second action potential from this fiber arrived at the recording electrode 59.6 ms after the first action potential, which is 0.4 ms less than the stimulus interval; this represents a decrease in conduction latency of 0.3% (or an increase in conduction velocity of 0.3%). The 24 sympathetic efferent fibers tested with twin pulse stimulation exhibited a mean decrease in conduction latency of 0.61 + 0.16%. The change in conduction latency between the first and second action potential we call the ‘‘twin pulse difference.’’ A negative Conduction in Small Fibers twin pulse difference corresponds to a decrease in conduction latency for the second action potential or an increase in con- duction velocity (i.e., speeding). The normalized twin pulse latency difference that is plotted in the figures was computed as the difference between the latencies of the first and second AP divided by the latency of the first AP. As shown in Fig. 3A (circles), the twin pulse difference was correlated with conduction velocity (R2 = 0.63, p,0.001); the greatest supra-normal conduction was observed at the slowest conduction velocities. Afferent Fibers Exhibit Sub-Normal Conduction In response to twin pulse stimulation at the sciatic nerve, all afferent fibers exhibited a slowing of conduction (i.e., a positive twin pulse difference); the second action potential was conducted at a slower conduction velocity than the first action potential. For the examples in Fig. 2C, the stimulus interval was 50 ms. In this recording, action potential waveform numbers 1, 2, and 3 (at latencies of 234.1, 247.6, and 251.7 ms, respectively) were positively identified as belonging to dorsal root afferent fibers. For the fiber represented by waveform # 2, the second action potential arrived at the recording electrode 52.2 ms after its first action potential, which is 2.2 ms greater than the stimulus interval; this represents an increase in conduction latency of 0.9% (or a decrease in conduction velocity of 0.9%). The other two afferent fibers in this recording (waveform # 1 and 3) also exhibited a longer latency for the second action potential (by 0.9 and 1.0%, respectively). The 48 afferent fibers tested with twin pulse stimulation exhibited a mean increase in conduction latency of 1.39 + 0.11% which was significantly different from that observed for sympathetic efferent fibers (p,0.001). As shown in Fig. 3A (triangles), the twin pulse difference was weakly correlated with conduction velocity (R2 = 0.19, p,0.01). Notably, the data for the afferent and efferent fibers appeared to form two clusters that were essentially non-overlapping (Fig. 3A). Activity-Dependent Slowing in Sympathetic Efferent Fibers A train of 360 stimuli (2 Hz for 3 min) was used to investigate activity-dependent slowing of conduction [17]. For the example sympathetic efferent fiber in Fig. 2A, the conduction latency increased during the first 100 pulses, but approached a plateau level for subsequent pulses (Fig. 2B); the final increase in latency was 12.9 ms which corresponded to an 8.7% increase in conduction latency. The 9 efferent fibers studied with this protocol exhibited a similar time course for their increase in conduction latency (Fig. 4B); the mean increase in latency at the end of the protocol was 8.6 + 0.6% (Fig. 4C). We did not observe a reversal of the conduction latency increase near the end of the 3 min stimulation as has been reported in human efferent fibers recorded in distal nerves [20]. Activity-Dependent Slowing in Afferent Fibers The response of a typical afferent fiber to the 2 Hz, 3 min stimulus train is illustrated in Fig. 2D. For this fiber (corresponding to waveform # 2 in Fig. 2C), the conduction latency increased continuously throughout the stimulation; the final increase in latency was 60.7 ms which corresponded to a 24.6% increase in conduction latency. The other two afferent fibers in this recording (waveform # 1 and 3 in Fig. 2C) also exhibited pronounced activity-dependent slowing (by 22.7 and 25.0%, respectively). The 23 afferent fibers studied with this protocol in centrifugal recordings exhibited two different types of slowing. For 17 afferent fibers (labeled as ‘‘big slowers’’ in Fig. 4A), the conduction latency PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9076