centripetal and centrifugal recordings from peripheral nerves, we found that activity-dependent slowing of conduction to repeated electrical stimulation was typically less in sympathetic fibers than in nociceptive afferent fibers. An important novel finding was that sympathetic efferent fibers in cutaneous nerves exhibit a speeding of conduction to a single pair of electrical stimuli separated by 50 ms; the second action potential was conducted faster than the first unconditioned action potential. This was never observed in unmyelinated afferent fibers. This difference provides a criterion by which sympathetic efferents and unmyelinated afferents can be differentiated in peripheral nerve recordings. Unmyelinated Nerve Fibers Exhibit Different Patterns of Conduction Velocity Slowing upon Repetitive Electrical Stimulation In centrifugal recordings from sciatic nerve fibers that were positively identified as afferent or efferent using collision techniques, we found different conduction slowing patterns in response to 2 Hz repetitive stimulation (3 min). Afferent fibers exhibited two distinct patterns: one afferent group (‘large’ slowers) slowed by $20% of the initial conduction velocity, whereas a second afferent group (‘small’ slowers) slowed significantly less (,7%). Such a dichotomy is in agreement with previous studies in rat [7,22,6] and human [17,10,23] in which the first group likely consists of nociceptive afferents, and the second group of ‘cold’ fiber afferents. The fibers in this study that were positively identified as sympathetic efferents exhibited a slowing that was similar to the second group of afferents; this finding is consistent with observations made in microneurographic recordings in human [20]. There are, however, differences between previous studies and the present study: 1) In recordings from human peripheral nerve, about 35% of fibers showed pronounced slowing ($30%) [17], but in the present study less than 25% of unmyelinated afferent fibers showed such slowing in centrifugal recordings. However, we applied electrical stimuli to the proximal nerve trunk and not to cutaneous peripheral terminals which have been suggested to contribute mainly to conduction slowing. Thus a difference in stimulation site may account for these differences. 2) We did not observe unmyelinated afferents with a ‘type 39 slowing that have been hypothesized to represent low threshold C fiber afferents and that are characterized by only a small increase (,1%) in latency during ongoing stimulation (2 Hz, 3 min). However, the incidence of such afferents is small (,5–6%) [17,11], and they may have been missed in our centrifugal recordings. Previous investigators postulated that activity dependent hyperpolarization associated with the Na+/K+ pump accounts for conduction slowing in unmyelinated afferents during repetitive electrical stimulation [14,9,11]. However, blockade of Na+/K+ ATPase has recently been demonstrated to increase conduction slowing [12]. In addition, the degree of use-dependent slowing of conduction was not affected by blocking TTX sensitive currents with low doses of tetrodotoxin (TTX). Instead, the use dependent slowing was sensitive particularly to lidocaine and carbamazepine, the two sodium channel blockers that have been known for their greater effectiveness in blocking slow inactivated NaV1.8- than TTX sensitive channels. Based on these data the authors suggest that conduction slowing upon repetitive electrical stimulation is due to slow inactivation of TTX resistant sodium channels, especially NaV1.8, and that hyperpolarization induced by activity in Na+/K+ ATPase rescues sodium channels from inactivation [12]. Differences in activity dependent slowing are likely due to differences in sodium channel expression in axonal membranes of the different C fiber populations. For example, dorsal root Conduction in Small Fibers ganglion neurons, but not superior cervical ganglion neurons, express the TTX resistant sodium channel NaV1.8 [24] which has been suggested to underlie activity dependent slowing in nociceptive afferents because this channel more readily enters a slow inactivated state upon activation and recovers slowly from that inactivation [13,25]. Absolute Speeding Distinguishes Sympathetic Efferents from Unmyelinated Afferents In centrifugal recordings, we observed that sympathetic efferents, but not primary afferents, exhibited absolute speeding of conduction when tested with a single twin pulse (ISI 50–70 ms); the conduction velocity of the second action potential was faster than the first, naive action potential. Similarly, in centripetal recordings, absolute speeding of conduction to a single twin pulse was observed in nerve fibers that were unresponsive to mechan- ical, thermal or chemical stimuli. Because of these features it is likely that these unmyelinated fibers are sympathetic efferent fibers. In contrast, mechanosensitive fibers only showed slowing of conduction to a single twin pulse. In addition, mechanoinsensitive fibers that were responsive to chemical stimuli and hence were likely afferents, exhibited conduction slowing in response to a single twin pulse. Only a few chemosensitive, mechanoinsensitive afferents were studied in centripetal recordings, and we therefore cannot exclude the possibility that some groups of mechan- oinsensitive afferents (e.g. cold nociceptive afferents), which we did not encounter in centripetal recordings, will show absolute speeding to twin pulse stimulation. This possibility, however, seems unlikely, since none of the fibers identified as afferents in the centrifugal recordings showed absolute speeding to twin pulses stimulation. Absolute speeding following a single twin pulse appears to be a distinct feature of mechanoinsensitive, efferent nerve fibers. Similar observations have recently been made in recordings from peripheral nerve fibers in pig (O. Obreja and M Schmelz, personal communication). Furthermore, absolute speed- ing to a single twin pulse (ISI 50 ms) is also likely to be present in sympathetic nerve fibers of human and rat since recovery cycles of conduction velocity in sympathetic nerve fibers in these species show an increase in conduction velocity at interspike intervals below 150 ms regardless of mean stimulation frequency [26,11]. Following an action potential, the axonal membrane of unmyelinated nerve fibers undergoes time dependent excitability changes as originally proposed by Barrett and Barrett [27] and supported by recent findings in human [14,9] and rat [11]; this after-potential in unmyelinated axons appears to reflect a residual charge on the membrane which decays exponentially through the membrane resistance-capacitance (RC) circuit. The size and polarity of this charge is dependent on the voltage-dependent gating mechanism of ion channels. For example, a depolarizing after-potential occurs if the outward potassium movement does not quite compensate for the inward sodium movement leading to excess intracellular sodium; this would lead to a ‘‘hyperexcitability’’ of the membrane and a speeding of conduction as seen in the sympathetic fibers. A hyperpolarizing after-potential occurs when the outward potassium current exceeds the inward sodium move- ment; this would lead to a ‘‘hypoexcitability’’ of the membrane and a slowing of conduction as seen in the afferent fibers. Our findings are consistent with a previous study [28] reporting a ‘positive’ (i.e. hyperpolarizing) afterpotential in unmyelinated vagal afferents and a ‘negative’ (i.e. depolarizing) afterpotential in sympathetic fibers of the hypogastric nerve. The difference between afferents and efferents (for a single twin pulse) may reflect a difference in the potassium/sodium channels that are expressed in afferents and sympathetic efferents. For example, NaV1.8 is not expressed in PLoS ONE | www.plosone.org 9 February 2010 | Volume 5 | Issue 2 | e9076