Figure 3. Twin pulse stimulation reveals two distinct popula- tions. A) Afferent and efferent fibers have different twin pulse responses. Following electrical twin pulses (50–70 ms ISI) at the sciatic nerve, all afferent fibers (red triangles, n = 48) exhibited an increase in conduction latency for the second action potential (AP). In sympathetic efferent fibers (blue circles, n = 24) the change in conduction latency was dependent on the initial conduction velocity (R2 = 0.63); slower fibers showed a decrease in latency (i.e., speeding of conduction) whereas faster fibers demonstrated a modest increase in latency. The data clusters are almost non-overlapping. A negative value for twin pulse latency difference means that the second AP has a shorter latency than the first AP. B) The initial twin pulse response does not predict the amount of activity-dependent slowing in the afferents. The ‘‘big slowers’’ and ‘‘small slowers’’ in response to the repeated stimulation paradigm (Fig. 4) have a similar twin pulse difference but different conduction velocities. The data are from all fibers in which the activity- dependent slowing protocol (2 Hz, 3 min) was performed. C) Mechani- cally-sensitive and -insensitive fibers respond differently to Conduction in Small Fibers twin pulse stimulation. In a separate series of experiments, the response to electrical stimulation at peripheral terminals was examined. Mechanically sensitive nociceptors (triangles) exhibited twin pulse slowing. Most mechanically-insensitive fibers behaved like sympathetic fibers (blue circles) and exhibited twin pulse speeding. In ten mechanically-insensitive fibers capsaicin and/or histamine were injected into the electrical receptive field; three responded (pink circles) and were likely afferents; seven were unresponsive (yellow circles) and are presumed sympathetic fibers. One mechanically-insensitive fiber (green circle) not tested with chemicals behaved like an afferent to the repeated twin pulse paradigm (see Fig. 6). Two mechanically-insensitive fibers (open circles) not tested with chemicals or repeated twin-pulses exhibited twin-pulse slowing and may therefore be afferents. doi:10.1371/journal.pone.0009076.g003 speeding was similar to that observed for the sympathetic efferent fibers (Fig. 3A) in the centrifugal recordings. To test whether these mechanically-insensitive fibers were afferents, we injected histamine (10 mg/10 ml) and/or capsaicin (10 mg/10 ml) into the electrical receptive field in 10 of these fibers. Seven did not respond to histamine or capsaicin, but three of these mechanically-insensitive fibers did respond and are likely mechanically-insensitive afferents. The responses of one of these fibers (labeled AC36 in Fig. 3C) to the intradermal injection of histamine and capsaicin are shown in Fig. 5. Notably, the three mechanoinsensitive fibers responsive to histamine/capsaicin (pink circles in Fig. 3C) exhibited twin-pulse slowing comparable to that seen in the mechanically-sensitive afferents (red trian- gles in Fig. 3C), whereas the seven chemically unresponsive fibers (yellow circles in Fig. 3C) exhibited twin-pulse speeding comparable to the presumed sympathetic fibers (blue circles in Fig. 3C). While conduction velocities of different groups of fibers overlapped, differences in latencies after twin pulse stimulation in mechanoinsensitive afferents and presumed sympathetic fibers did not, i.e. the change in latency following twin pulse stimulation differentiates mechanoinsensitive afferents from sympathetic efferent fibers. Supra-Normal Conduction Is Dependent on Stimulus Interval In centripetal recordings we used the 60 twin pulses protocol to investigate the relationship of activity-dependent slowing and relative speeding of conduction. A subset of fibers was tested with twin pulse stimulation where pulses of twin stimuli were separated by a fixed stimulus interval (10, 20, 50, 100, 200 or 500 ms). In response to the first twin pulse, two outcomes were observed: 1) a negative twin pulse difference that indicates supra-normal conduction of the second action potential relative to the first action potential, or 2) a positive twin pulse difference indicating a slowing of conduction. Based on our centrifugal recordings, a fiber was presumed to be a sympathetic efferent if it showed a negative twin-pulse difference to the first twin pulse at 50-70 ms stimulus interval and was unresponsive to noxious mechanical or chemical stimuli. Units responsive to mechanical stimuli were categorized as MSA fibers. For the presumed sympathetic fibers, a speeding of conduction was observed to the first twin pulse over stimulus interval range of 20 to 100 ms, with slowing of conduction at ISIs of 10 ms and 500 ms (filled circles in Fig. 6A). The greatest speeding occurred at the 50 ms ISI. For the MSA fibers, slowing of conduction (i.e., positive twin pulse difference) was observed to the first twin pulse over the entire range of stimulus intervals (filled circles in Fig. 6B). The largest twin pulse slowing (6.2 + 0.3%) occurred for the10 ms ISI, and the amount of slowing decreased as the ISI increased. PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9076