First, we investigated whether single C-fibers could be activated by three different chemicals: Potassium chloride (KCl), gamma-aminobutyric acid (GABA), and capsaicin; due to the high intracellular chloride concentration of peripheral neurons an excitatory effect of GABA is expected. Immunohistochemistry revealed the co-expression of TRPV1 and GABA_A receptors in the some dorsal root ganglion (DRG) neurons (Figure 1A). Patch-clamp recording showed that GABA and capsaicin evoked inward currents in the same DRG neuron (Figure 1B) but that not all DRG neurons were responsive to both chemicals (7 of 28 total neurons responded for both GABA and capsaicin). In ex vivo recordings, 27 C-fibers were stimulated with the three chemicals in succession, with wash-out periods in between. Three fibers responded to all stimulants with identical AP shapes and sizes (Figure 1C, Supplementary Tables 1, 2).

It is known that C-fiber activation by KCl or capsaicin results in nociception. However, the sensation evoked by GABA is not reported despite the activation of C-fibers (Feltz and Rasminsky, 1974; Deschenes et al., 1976; Carlton et al., 1999). To test whether the application of GABA evoked a nociception, we performed behavioral tests for each chemical. Mouse hindpaw movements indicating pain, i.e., lifting/guarding, flinching/shaking, licking, and walking (Kawasaki-Yatsugi et al., 2012), significantly increased after subcutaneous injection of capsaicin or KCl (Ps < 0.001 vs. vehicle). On the other hand, GABA induced no significant increase (P = 0.693; Figure 1D), indicating that GABA could not induce nociception.

To further explore nociceptive information encoded in the activation of primary afferent C-fibers, ex vivo single-fiber recordings were conducted with each of the three chemicals separately (Figure 2). Analysis of the spike counts in ex vivo single-fiber recordings from the saphenous nerve showed that all three chemicals caused significant increases in the number of APs compared with control periods (Ps < 0.001; Figures 2D–F). In addition, there was no significant difference in the number of APs evoked by the three stimulants (P = 0.925; Figure 2G). Another measure of discharge rate is the instantaneous frequency (Lánský et al., 2004) and it is claimed that a discharge rate exceeding some threshold is associated with nociceptive behavior (Adriaensen et al., 1980). As shown in Figures 2A–C, the distribution of the instantaneous frequencies of GABA responses (mean: 1.72 Hz) did not differ from that of the KCl responses (mean: 1.25 Hz) while that of capsaicin responses (mean: 17.49 Hz) was much higher (Figures 3A–D).

As neither the number of APs nor the instantaneous frequencies could differentiate GABA responses from the other two responses, we considered the possibility that the differences might be temporally encoded in the spike trains. Visual inspection indicated that the most pronounced difference of GABA responses from the other responses lay in the repeated short bursts present in the GABA responses, we posited a fundamental unit of temporal discharge pattern to be three consecutive spikes (referred to as a spikelet hereafter), as this unit contained the minimum ISI pattern that could include both short ISIs within bursts and longer ISIs between bursts. In contrast to the results of spike counts or instantaneous frequency, GABA responses had a longer mean spikelet length (time elapsed from the first to the last spike in a spikelet) than those to the other chemicals (Figures 3E–H). The longer GABA spikelet lengths were primarily caused by a high frequency of relatively long spikelets (Figure 3G), which indicated that long and short ISIs were repeated in succession across bursts during a GABA response. To also measure asymmetry of two ISIs in a spikelet due to the intersection of long and short ISIs, we evaluated the distribution of absolute spikelet regularity (the ratio of difference between two consecutive ISIs to spikelet length) of each group and found that the regularity of GABA spikelets was significantly lower than those of KCl or capsaicin spikelets (Figures 3I–L). Both the length and the regularity of spikelets clearly distinguished the temporal pattern of GABA responses from the others, and this might be associated with the lack of nociceptive behavior from GABA.

We also analyzed the joint distribution of the two temporal features, the length and the regularity of spikelets and created a two-dimensional (2D) joint distribution map of spikelets for each group (Figure 4). The maps of each group could be distinguished from the others. KCl frequently generated temporal patterns with regular spikelets of a moderate length, whereas GABA tended to generate more irregular and longer spikelets. Capsaicin tended to generate short and regular spikelets (Figures 4B–D).

The three distribution patterns were clearly different, as the k-nearest neighbor classification scheme correctly discriminated between them with classification accuracy of 79.7% (55 of 69 C-fiber responses classified correctly), which was significantly higher than chance (binomial test, P < 0.001; Devroye et al., 1996). In addition, nine individual maps of the three C-fiber responses obtained from the successive chemical tests in the above section were also perfectly classified into KCl, GABA, and capsaicin using a classifier based on the set of three separate spike train datasets generated by the individual chemical stimuli (Supplementary Figure 1). This indicates that C-fibers may encode information concerning the specific receptors that have been stimulated by means of unique temporal patterns.

In an attempt to estimate the effect of the temporal summation of discharge patterns on the nociceptive level, a computational model was designed using the information of spikelet length and regularity. The model was applied to the C-fiber responses with over 20 spikes during the stimulation period to estimate the nociceptive level through the temporal summation of spikelet information. As a result, the detection rate of nociception by the model was 66.7% (14 of 21) for KCl, 17.4% (4 of 23) for GABA, and 88.9% (16 of 18) for capsaicin, respectively. This result indicated that the model could properly translate C-fiber spiking patterns, represented by spikelet length and regularity, into the nociception level in response to different chemical stimuli.

We examined whether changes in pathological conditions could influence discharge patterns in C-fibers and subsequent nociceptive behavior (Figure 5). We conducted behavioral tests for GABA and capsaicin in mice subjected to chronic constriction injury (CCI; sciatic nerve cuffing). Compared with the controls, the number of hind paw movements were increased by both GABA and capsaicin (P < 0.01; Figure 5A), indicating that GABA induced a nociceptive behavior when exposed to a CCI.

The GABA and capsaicin discharge patterns were investigated using ex vivo single-fiber recordings from the sural nerves of CCI mice. First we found that the number of APs increased in response to GABA or capsaicin compared with control periods (Figures 5D,E) but there were no significant differences in the number of GABA- or capsaicin-induced APs in the CCI mice compared with the naïve mice (Supplementary Figure 2). The 2D joint distribution map for capsaicin in the CCI mice (Figure 6B) was closely akin to that in the naïve mice (Figure 4D). However, the GABA map in the CCI mice (Figure 6A) appeared to be dissimilar from that in naïve mice (Figure 4C). This suggested that the discharge pattern for a specific receptor in C-fibers could be influenced by the pathological conditions. Specifically, the short and regular spikelets, which were abundant in the capsaicin spike trains, became more frequent in the GABA spike trains of the CCI mice (Figure 6C). The abundance of the short and regular spikelets characterizing the capsaicin and CCI-induced GABA discharge patterns implied that the generation of pain might be related to the presence of high frequency continuous spikes in the afferent C-fibers. K-nearest neighbor classification analysis revealed that the binary classifier that had been trained using the GABA and capsaicin data from naïve mice had little difficulty in discriminating the novel GABA data from naïve mice but made many more errors (nearly 50% of the time) in discriminating the GABA data from the CCI mice, indicating that the GABA discharge patterns in the CCI mice were less distinguishable from the capsaicin discharge patterns (Figure 6D). Similarity analysis based on Kullback–Leibler (KL) divergence also revealed that the GABA pattern in the CCI mice was more similar to the capsaicin pattern (Figure 6E).

All experiments were conducted according to the guidelines of the Animal and Plant Quarantine Agency of Korea for the care and use of laboratory animals, and the study was approved by the Institutional Animal Care and Use Committee of Hanyang University (HY-IACUC-13-037A). Male C57BL/6 mice (8 weeks old) were used throughout the study.

First, the effect of the studied chemicals on nociceptive behavior was investigated. Behavioral changes were observed in 46 mice after subcutaneous injection of one or other of the chemical stimulants, KCl (n = 10), GABA (n = 10), or capsaicin (n = 8), or a control, saline (n = 10) or vehicle (n = 8).

Next, to examine the sensitivity of single C-fibers to multiple chemicals, ex vivo recordings were made with 14 fibers from another group of mice. Each fiber was tested by application of all three chemicals in succession, with a wash-out period between each application. The average conduction velocity of the fibers in the sensitivity test was 0.65 ± 0.17 m/s. In addition, the effects of the three chemicals (KCl, n = 25; GABA, n = 23; capsaicin, n = 21) on the neural response were tested separately, with each fiber exposed to only one of the chemical stimulants. To exclude extraneous effects, the mice used for the behavioral tests were not used for ex vivo recording. For a pathological model, 42 mice were subjected to CCI by sciatic nerve cuffing. Hypersensitivity to mechanical and heat stimuli was verified 12–15 days after surgery. Of the 42 CCI mice 26 were used for the behavioral test (response to vehicle, n = 8; GABA, n = 9; capsaicin, n = 9). Fibers for ex vivo recording (response to GABA, n = 36 or capsaicin, n = 46) were obtained from the remaining 16 mice (Figure 7).

The CCI model was used for behavioral tests based on hindpaw movement (response to stimulus to the plantar surface of the foot) as well as ex vivo recording from the hairy skin under pathological conditions. Surgery was performed under brief isoflurane anesthesia. The main branch of the left sciatic nerve was exposed and a cuff of PE-20 polyethylene tubing (0.38 mm internal diameter, 1.09 mm external diameter; Harvard Apparatus) of standardized length (2 mm) was applied (Benbouzid et al., 2008). The shaved skin layer was closed using sutures. Compared with baseline, the mechanical and heat thresholds were significantly reduced following sciatic nerve cuffing on postoperative days 12–15 (Wilcoxon signed rank test, Ps < 0.001), indicating neuropathic pain. The left sural nerve, which was affected by the CCI, was used for ex vivo recording.

Mice were acclimated to testing cages containing either a stainless steel mesh (for mechanical withdrawal responses) or a heat-tempered glass floor (for spontaneous pain behavior and heat withdrawal latency) for 2 h per day for at least 5 days before testing. All behavioral tests were performed after at least 1 h acclimation on the day of the experiment.

Mice were killed using CO₂ inhalation and the hairy skin of the hindpaw innervated by the saphenous or sural nerve was dissected after the hair on the leg was clipped. For recording from the CCI model mice, only the left sural nerve, which was impacted by the cuffing of the left sciatic nerve, was used. Attached connective tissue, muscle, and tendon were removed. The organ bath consisted of two chambers separated by an acrylic-based wall. The larger perfusion chamber was continuously superfused with a SIF (107.8 mM NaCl, 3.5 mM KCl, 0.69 mM MgSO₄·7H₂O, 26.2 mM NaHCO₃, 1.67 mM NaH₂PO₄·2H₂O, 9.64 mM C₆H₁₁NaO₇, 5.55 mM glucose, 7.6 mM sucrose, 1.53 mM CaCl₂·2H₂O) saturated with a mixture of 95% O₂ and 5% CO₂ (Bretag, 1969). The temperature of the bath solution was maintained at 31 ± 1°C. After dissection, the preparation was placed with the epidermal side down. The nerves attached to the skin were drawn through one small hole to the smaller second chamber, which was filled with paraffin oil. The nerve was placed on a fixed mirror, the sheath was removed, and nerve filaments repeatedly teased apart to allow single-fiber recordings to be made using gold electrodes, one for recording and the other for reference. The reference electrode was grounded to the perfusion chamber. Signals from single nociceptive afferent fibers were recorded extracellularly with a differential amplifier (DP 311; Warner instruments). Amplified signals were sent to an oscilloscope and an audio monitor and sampled at 33 kHz, then transferred to a computer by a data acquisition system (DAP5200a; Microstar Laboratories, Inc.). APs collected on the computer were analyzed off-line using the window discrimination feature of the software (Dapsys 8; Bethel University, http://dapsys.net/). Copper blocks were connected to common ground as a reservoir of current to prevent excessive noise. The entire setup was based on a study by Zimmermann et al. (2009). The conduction velocity of the axon was determined by monopolar electrical stimulation through a low impedance electrode (CBJPL75; FHC Inc.). The supramaximal square-wave pulses (0.2–2 ms duration, 0.5 Hz) were delivered at the mechanosensitive site of a receptive field using an electrical stimulator (SD9; Grass Technologies). The distance between the receptive field and the recording electrode (conduction distance) was divided by the latency of the AP. A single C-fiber was selected on the basis of the conduction velocity (slower than 1.2 m/s); fast conducting A-fibers were excluded. The primary search strategy was mechanical stimulation by a fire-polished glass rod targeting mechanosensitive fibers.

A histogram of instantaneous frequencies was constructed to show the empirical distribution of instantaneous frequencies for each chemical stimulus. For each chemical, all the instantaneous frequency values were divided into 10 equal-length (0.8 Hz) bins from 0–0.8 to 7.2–8 Hz. Frequency values >8 Hz were included in the last bin.

Given a spike train of N spikes, a spikelet s was defined as a set of three consecutive spikes, (1)s=(tn,tn+1,tn+2), where n = 1, …, N−2. We characterized spikelets in terms of two simple and basic parameters: length and regularity. Spikelet length L was defined as the time elapsed from the first to the last spike, (2)L=(tn+2−tn). Spikelet regularity was defined as the ratio of increment of two consecutive ISIs to spikelet length, (3)Ψ=2(tn+2−tn+1)/(tn+2−tn). Note, that spikelet regularity ranges from 0 to 2. Finally, we identified every spikelet in the spike train, allowing overlapping of two spikes between successive spikelets and calculated the length and regularity of each (i.e., there are N–2 spikelets in a train of N spikes where N > 2).

Having measured spikelet length and regularity from the spike train generated in response to a particular chemical, we built histograms of each parameter. The values of spikelet length were divided into 10 bins in a logarithmic scale with base 2 from 0 to 8 s: 0–0.157 s, 0.157–0.250 s, 0.250–0.397 s, 0.397–0.630 s, 0.630–1.000 s, 1.000–1.587 s, 1.587–2.520 s, 2.520–4.000 s, 4.000–6.350 s, and 6.350 s to the maximum. The maximum length was set as 8 s in our analysis, and longer lengths were included in the last bin. The absolute values of spikelet regularity, |Ψ|, were divided into 10 equal-length bins from 0–0.1, 0.1–0.2, …, 0.9–1.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.