Selective activation of primary afferent fibers evaluated by sine-wave electrical stimulation

Abstract

Transcutaneous sine-wave stimuli at frequencies of 2000, 250 and 5 Hz (Neurometer) are thought to selectively activate Abeta, Adelta and C afferent fibers, respectively. However, there are few reports to test the selectivity of these stimuli at the cellular level. In the present study, we analyzed action potentials (APs) generated by sine-wave stimuli applied to the dorsal root in acutely isolated rat dorsal root ganglion (DRG) preparations using intracellular recordings. We also measured excitatory synaptic responses evoked by transcutaneous stimuli in substantia gelatinosa (SG) neurons of the spinal dorsal horn, which receive inputs predominantly from C and Adelta fibers, using in vivo patch-clamp recordings. In behavioral studies, escape or vocalization behavior of rats was observed with both 250 and 5 Hz stimuli at intensity of approximately 0.8 mA (T5/ T250), whereas with 2000 Hz stimulation, much higher intensity (2.14 mA, T2000) was required. In DRG neurons, APs were generated at T5/T250 by 2000 Hz stimulation in Abeta, by 250 Hz stimulation both in Abeta and Adelta, and by 5 Hz stimulation in all three classes of DRG neurons. However, the AP frequencies elicited in Abeta and Adelta by 5 Hz stimulation were much less than those reported previously in physiological condition. With in vivo experiments large amplitude of EPSCs in SG neurons were elicited by 250 and 5 Hz stimuli at T5/ T250. These results suggest that 2000 Hz stimulation excites selectively Abeta fibers and 5 Hz stimulation activates noxious transmission mediated mainly through C fibers. Although 250 Hz stimulation activates both Adelta and Abeta fibers, tactile sensation would not be perceived when painful sensation is produced at the same time. Therefore, 250 Hz was effective stimulus frequency for activation of Adelta fibers initiating noxious sensation. Thus, the transcutaneous sine-wave stimulation can be applied to evaluate functional changes of sensory transmission by comparing thresholds with the three stimulus frequencies.

Figure 1

Representative firing patterns of C (A), Aδ (B) and Aβ (C) neurons in response to 2000 Hz sine-wave stimulation. In this and subsequent figures 2 and 3, the stimulus intensities were shown above the traces. Action potentials recorded from three types of afferent fibers were shown on the right of the traces. The frequencies of APs were plotted against the stimulus intensity (D). In this and subsequent figures of 2 and 3, ○: C fiber (n = 15), △: Aδ fiber (n = 18), □: Aβ fiber (n = 20).

Figure 2

Representative firing patterns of C (A), Aδ (B) and Aβ (C) neurons in response to 250 Hz sine-wave stimulation. The frequencies of APs were plotted against the stimulus intensity (D).

Figure 3

Representative firing patterns of C (A), Aδ (B) and Aβ (C) neurons in response to 5 Hz sine-wave stimulation. The frequencies of APs were plotted against the stimulus intensity (D).

Figure 4

EPSCs in SG neurons occurring spontaneously and in response to pinch stimulation in vivo (A). Pinch stimulation was applied to the skin of the hind limb. EPSCs indicated by arrowheads and pinch-evoked EPSCs were shown an expanded time scale in B and C, respectively.

Figure 5

EPSCs in SG neurons evoked by 5 (A), 250 (B) and 2000 (C) Hz transcutaneous sine-wave stimuli. In A-C, the stimulus intensities were shown above the traces. Lower two records in A were shown in an expanded time scale. Relative frequencies of large amplitude of EPSCs evoked by the stimuli at intensities of T5, T250 or T 2000 (D). *P < 0.05, **P < 0.01.

Figure 6

EPSCs in SG neurons recorded in the presence of TTX (1 μM, A) or CNQX (20 μM, B).

Figure 7

Schematic diagram of activation of afferent fibers with sine-wave stimuli. The pie showed the number of three types of rat DRG neurons as reported by Djouhri et al [32]. The height represented normalized AP frequencies produced by sine-wave stimuli in the three types of fibers in our study relative to those by natural stimuli reported previously [31]. This figure shows the selectivity of the sine-wave stimuli to activate afferent fibers as described in 'Discussion'.