Immediately following an action potential (i.e., within 500 ms), the axonal membrane of unmyelinated nerve fibers undergoes time dependent excitability changes [14]. The refractory period may be followed by a phase of hyperexcitability which then is succeeded by a period of hypoexcitability. Similar to activity dependent slowing, post excitatory excitability changes appear to differ between classes of unmyelinated nerve fibers [9,11]. In peripheral nerve recordings of cutaneous terminals, it is difficult to distinguish mechanoinsensitive afferents from sympa- thetic efferents since neither respond to mechanical stimuli. In human microneurography, sympathetic maneuvers (e.g., valsalva) can be used to induce a response in some sympathetic fibers, but such procedures do not distinguish afferent and efferent fibers in preparations in which axons are cut and neuronal activity is recorded from the peripheral axonal process distal to the cut. Moreover, the adequate stimulus by which mechanoinsensitive afferents could be activated is often unknown, and fibers are classified as ‘sympathetic’ by a lack of response to thermal or chemical stimuli. We therefore sought a criterion based on conductive properties that could be used to differentiate sympathetic from afferent fibers. Using centrifugal recordings in which sympathetics and afferents could be positively identified by stimulation of the sympathetic chain or the dorsal root, respectively, we found that sympathetic efferent fibers exhibited an absolute speeding of conduction to a single pair of electrical stimuli separated by 50 ms; this was never observed in primary afferent fibers. This presence of absolute speeding provides a criterion by which sympathetic efferents can be differentiated from primary afferents. Methods Ethics Statement Experiments were approved by the Animal Care and Use Committee of the Johns Hopkins University (protocol PR06M396). Experiments were performed in accordance with Animal Welfare Act regulations and the USPHS Policy on Humane Care and Use of Laboratory Animals. General Procedures Monkeys (Macaca fascicularis, 4–6 kg) were initially sedated by intramuscular ketamine (12 mg/kg with 0.04 mg/kg atropine) and anesthesia was induced by an intravenous bolus injection of sodium pentobarbital (6 mg/kg) and maintained by constant infusion (4–6 mg/kg/hr) thereof. Hydration was maintained with a continuous infusion of 5% dextrose in physiological saline solu- tion. An intramuscular injection of penicillin (600,000 U) was given for prophylaxis against infection. Intubation was performed, and animals were paralyzed with an intravenous dose of pancur- onium bromide (0.1 mg/kg) every 2 hours or when necessary. Ventilation was adjusted throughout the experiment to maintain the expired pCO2 within physiological range (i.e., 35–45 mmHg). Heart rate was monitored by an electrocardiogram to ensure an adequate level of anesthesia. Supplemental doses of pentobarbital were administered if the heart rate increased by more than 10% upon application of noxious stimuli. Core temperature was monitored via a rectal thermometer and maintained near 38uC with feedback- controlled circulating water heating pads. Teased-fiber recording techniques [15] were used to record from single C fibers in peripheral nerves. Briefly, small filaments were cut from the nerve and placed on a dissecting platform. A smaller strand was dissected from the filament and looped around a silver-wire recording electrode. A nerve-trunk electrode was used to electrically stimulate the nerve to reveal the fibers on the recording electrode. Conduction in Small Fibers In experiments on 5 animals, a laminectomy over the lumbar spine was performed to gain access to the lumbar dorsal roots. After exposure of the lumbar spinal cord, the skin of the back was sutured to a metal ring to form a pool that was filled with paraffin oil. The dura was then slit open along the length of the exposed spine, and multiple dorsal roots were carefully isolated for electrical stimulation. Electrical stimulation of the dorsal roots was performed using bipolar stimulation electrodes. The anato- mical level of the stimulated dorsal roots was not determined; however, stimulation of multiple roots always induced centrifugal activity that could be recorded in the superficial peroneal nerve. In separate experiments in 4 other animals, the lumbar sympathetic chain was exposed through a retroperitoneal, dorso- lateral approach as has been described previously in rat [16]. The ramus communicans between neighboring sympathetic ganglia was positioned on a bipolar stimulation electrode allowing stimulation of the sympathetic chain. The exact anatomical level of the sympathetic ganglia at which stimulation was performed was not determined. Animals undergoing laminectomy or exposure of the lumbar sympathetic chain were sacrificed at the end of the recordings by an overdose of pentobarbital. Centrifugal Recordings A schematic of the electrophysiological preparation is shown in Fig. 1A. Action potential activity was recorded in single C fibers in a cutaneous nerve (i.e, the superficial peroneal or sural nerve). The latencies of centrifugally propagating action potentials were obtained following electrical stimulation of the sciatic nerve. Bi- polar stimulation electrodes were also placed on the dorsal root or, in separate experiments, the sympathetic chain. Collision techni- ques 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 (see below). Only unmyelinated sciatic nerve fibers that were positively identified as efferent or afferent were studied. The conduction distance was measured along the course of the nerve from the recording electrode to the sciatic nerve electrode. The conduction velocity was calculated by dividing this distance by the conduction latency obtained from the sciatic nerve electrode with intensities at least 1.5 times threshold for excitation. Fibers with a conduction velocity of ,1.5 m/s were regarded as C fibers. Conduction velocities were also determined from the latencies following stimulation of the dorsal root or sympathetic chain electrode. Collision technique to identify positively afferent and sympathetic efferent fibers. The current intensity of the electrical stimulus (1/4 Hz) at the dorsal root (or sympathetic chain) was increased until an action potential waveform at a fixed latency was observed at the peroneal nerve recording electrode. Then, a simultaneous stimulus was applied to the sciatic nerve electrode, and the current strength increased until the action potential waveform initiated from the dorsal root (or sympathetic chain) disappeared. At this current, an action potential with a waveform identical to that initiated from the dorsal root (or sympathetic chain) always appeared at a shorter latency. This ‘short latency’ action potential had been initiated at the sciatic nerve stimulation electrode and had propagated both centrifugally towards the recording electrode and centripetally towards the dorsal root (or sympathetic chain). The centripetally propagating action potential collided with the centrifugally propagating action potential initiated at the dorsal root (or sympathetic chain) thereby preventing this action potential from being conducted towards the recording electrode. This ‘collision’ provided positive evidence PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9076