Figure 1. Schematic of electrophysiological preparation. A) Teased fiber techniques were used to record action potential activity in single C fibers in the peroneal nerve. The latencies of centrifugally propagating action potentials were obtained following electrical stimulation of the sciatic nerve. Collision techniques with stimulation at the sympathetic chain and the sciatic nerve or the dorsal root and the sciatic nerve were used to identify the peroneal nerve fiber as a sympathetic efferent fiber or a sensory afferent fiber. Only sciatic nerve fibers that were positively identified as efferent or afferent were studied. B) Schematic drawing of the transcutaneous electrical stimulation set up used in the centripetal recordings. Plastic wells were glued onto the skin over the receptive field of the unit under Conduction in Small Fibers study and filled with electrode gel. A silver wire was inserted into the gel and connected to an electrical stimulator. A needle inserted outside the receptive field but nearby served as a return electrode. In some cases, the receptive field was stimulated with two ball electrodes (spacing = 1 cm). In addition to the skin electrode, an electrode was also placed on the nerve trunk (N) between the peripheral receptive field and the recording electrode (R). C) A repeated twin-pulse stimulation protocol was used in the centripetal recording experiments. The first stimulus train consisted of 60 twin pulse stimuli; 50 ms separated the first and second pulse, and there was a 2 s interval between twin pulses. After a 2 min pause, additional twin pulse trains were delivered in which the interpulse interval was 10, 20, 100, 200, or 500 ms. doi:10.1371/journal.pone.0009076.g001 that an action potential waveform initiated from the sciatic nerve stimulus belonged to either a dorsal root afferent or a sympathetic efferent fiber. Experimental protocol. Following a stimulus free interval of at least 2 min, two protocols for the electrical stimulation of the sciatic nerve were used in experiments in which centrifugally conducted action potentials were recorded. Stimulus strength was set at 50% above threshold for activating the C fiber of interest. 1) Twin pulse conduction: A twin pulse stimulus at an interpulse interval of 50–70 ms was used to assess whether the conduction latency of the second action potential was shorter than the first (i.e., supernormal conduction or ‘‘speeding’’). 2) Activity-dependent slowing: A train of pulses (2 Hz for 3 min) was employed to investigate activity-dependent slowing of conduction. A similar protocol has been used by others to distinguish sympathetic efferent fibers from primary afferent fibers [17]. Centripetal Recordings In a separate series of 77 experiments in 21 animals, the conductive properties of fibers with peripheral, cutaneous terminals that were either mechanically-sensitive nociceptive afferent fibers or mechanically-insensitive fibers (efferent or afferent) were investi- gated. The receptive field of mechanically sensitive afferent fibers was mapped with von Frey probes. The electrical receptive field of mechanically-insensitive fibers was mapped using electrocutaneous stimulation. The electrical receptive field was defined as that region on the skin where the conduction latency decreased in discrete steps as the stimulus current increased [18]. In experiments where repeated twin pulse stimuli were delivered to the receptive field (see below), a 3-mm-diameter plastic well was glued to the skin in the receptive field and filled with electrolyte gel [19] to form a secure stimulating electrode (Fig. 1B). A coiled silver wire, positioned inside the well, served as the cathode. A stainless steel needle was inserted in the skin distal to the surface electrode and outside the receptive field. This electrode served as the anode. As the current was increased at this receptive field electrode, the latency of the recorded action potential decreased in discrete steps corresponding to excitation of the branching terminal structure at more proximal locations [19]. To insure that changes in action potential latency during our experimental protocol were not the result of hopping between different initiation sites in the peripheral terminal, we determined the largest current range over which a fixed latency was observed and set the stimulus current in the middle of this range (Fig. S1). Experimental protocol. All fibers were tested with at least one twin-pulse electrical stimulus (usually with an interstimulus interval of 50 ms). In a subset of fibers, electrical test stimuli consisted of a train of 60 twin stimuli with twin stimuli applied every 2 seconds. In a given train of twin stimuli, pulses of a twin stimulus were separated by a fixed interstimulus interval (ISI) PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9076