Conduction in Small Fibers Figure 4. Activity dependent slowing of afferent and sympa- thetic-efferent fibers. A) Afferents. In response to the train of 360 electrical pulses (2 Hz for 3 min), the afferent fibers exhibited two types of slowing in centrifugal recordings. The ‘‘big slowers’’ exhibited an increasing conduction latency throughout the stimulus train and reached a final conduction latency increase of greater than 20% (n = 17). The latency of the ‘‘small slowers’’ increased during the first 50 pulses but reached a plateau with a final conduction latency increase less than 10% (n = 6). B) Efferents. In centrifugal recordings, the sympathetic fibers (n = 9) looked like the small slowers and also had a final conduction latency increase less than 10%. C) Average activity dependent slowing for two types of afferents and for sympathetic efferents. The ‘‘small slowing’’ afferents and the sympathetic fibers exhibited similar activity dependent slowing. The ‘‘big slowing’’ afferent fibers were significantly different. Data were obtained from centrifugal recordings. doi:10.1371/journal.pone.0009076.g004 Activity-Dependent Slowing Potentiates Twin-Pulse Speeding Previous investigators have suggested that a relative speeding of conduction to twin pulse stimulation develops in C-fiber nociceptors in the presence of activity-dependent slowing [9]. To investigate this phenomenon, we delivered 60 twin pulses (Fig. 1C) to the cutaneous terminals of MSAs and presumed sympathetic fibers. Typical results from this protocol are illustrated in Text S1 (see also Fig. S2 and S3). In both classes of fibers, the repeated twin pulses led to the development of activity-dependent slowing of the first action potential of the twin pulse. For the presumed sympathetic fibers, Figure 5. Mechanically-insensitive afferent fiber responds to histamine and capsaicin. The C-fiber mechanically-insensitive afferent identified as AC36 in Fig. 3Cresponded to intradermal injection of histamine (10 mg/10 ml) and capsaicin (10 mg/10 ml). The response to both chemicals was greatest during the first 30 s and lasted about 10 min. Instantaneous frequency is plotted as a function of time following injection (each dot corresponds to time of an action potential). Note, there was no response during needle insertion or drug injection (shaded bar). doi:10.1371/journal.pone.0009076.g005 the activity-dependent slowing increased quickly (time constant = 27 s, Fig. S4B) and reached a plateau within the first 20 pulses. In contrast, the mechanically-sensitive afferents exhibited a continuous increase in activity -dependent slowing throughout the protocol (time constant = 64 s, Fig. S4B). Fig. 6A illustrates the average twin pulse data from the presumed sympathetic fibers. Several findings are noteworthy: 1) At the smallest ISI (i.e.,10 ms), only twin-pulse slowing was observed. With increasing number of twin stimuli, the twin pulse difference became smaller but remained positive throughout the stimulus train. 2) At the largest ISI (i.e., 500 ms), only twin-pulse slowing was observed. The magnitude of the twin pulse difference did not vary with increasing twin pulse stimuli. 3) Only small relative speeding was observed when twin pulses separated by 200 ms were applied. 4) For ISIs between 20 and 100 ms, speeding was present for the first twin pulse, and the amount of speeding increased as the number of twin pulses increased, but reached a plateau after about 20 twin pulses (see also Fig. S4C). 5) The largest twin pulse speeding occurred at the 50 ms ISI. This was true throughout the repeated stimulation protocol. The maximum speeding (3.1 + 0.5%) was observed at the end of stimulation at an ISI of 50 ms. The average data for all C-MSA fibers are shown in Fig. 6B. Several findings are noteworthy: 1) Regardless of the stimulus PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9076