Velocity recovery cycles of C fibres innervating human skin

Abstract

Velocity changes following single and double conditioning impulses were studied by microneurography in single human C fibres to provide information about axonal membrane properties. C units were identified as mechano-responsive (n = 19) or mechano-insensitive (12) nociceptors, cold-sensitive (8) or sympathetic fibres (9), and excited by single, double and triple electrical stimuli to the skin at mean rates of 0.25-2 Hz. The interval between single or paired (20 ms apart) conditioning stimuli and test stimulus was then varied between 500 and 2 ms, and recovery curves of velocity change against inter-spike interval constructed, allowing for changes in these variables with distance. All fibres exhibited an initial (4-24 ms) relative refractory phase, and a long-lasting (>500 ms) 'H2' phase of reduced velocity, attributed to activation of Na+/K+-ATPase. Mechano-responsive nociceptors exhibited an intermediate phase of either supernormality or subnormality, depending on stimulation rate. Mechano-insensitive nociceptors behaved similarly, but all were supernormal at 1 Hz. Sympathetic units exhibited only a long-lasting supernormality, while cold fibres exhibited a briefer supernormal and a late subnormal phase (H1), similar to A fibres. A pre-conditioning impulse doubled H2 and increased H1, but did not augment supernormality or the subnormality of similar time course. Like A fibre supernormality, these phenomena were explained by a passive cable model, so that they provide an estimate of membrane time constant. Nociceptor membrane time constants (median 110 ms, n = 17) were rather insensitive to membrane potential, indicating few active voltage-dependent potassium channels, whereas sympathetic time constants were longer and reduced by activity-dependent hyperpolarisation.

Figures

Figure 1. Latency tracking of recovery cycles with one and two conditioning impulses

A, filtered and inverted action potential recordings from a single cold-specific C fibre. Top traces, responses recorded on Qtrac channel 1, to test stimulus alone. Middle traces, responses on channel 2 to test stimulus preceded by conditioning stimulus. Bottom traces, responses on channel 3 to test stimulus preceded by conditioning and pre-conditioning stimuli, 20 ms apart. Left traces recorded at ≈0.5 min elapsed time (arrowed in B), with interstimulus interval 500 ms, showing small increase in latency for each conditioning stimulus. Right traces recorded at ≈3.5 min elapsed time, with interstimulus interval 50 ms, showing reduced latency with single conditioning stimulus. Inset, schematic indication of timing of test (T), conditioning (C) and pre-conditioning (P) stimuli. Horizontal lines are the moving ‘windows’ in which latencies are measured, which are automatically centred on the action potentials. B, latencies to test stimuli, plotted as function of elapsed time during the recording, with first minute expanded on left to show that adding conditioning stimuli while keeping mean stimulation rate constant does not affect baseline latency. L0, L1 and L2, latencies to responses in top, middle and bottom traces in A, i.e. latencies to responses to test stimuli preceded by zero, one and two conditioning stimuli respectively.

Figure 2. Recording of latency recovery cycle of sympathetic unit with pronounced supernormal period

Unit stimulated at mean rate of 1 Hz, with interstimulus interval of 1 s after single stimulus, 2 s after double stimulus and 3 s after triple stimulus. A, top, latencies to responses to unconditioned test stimulus (L0), to responses to test stimulus after single conditioning stimulus (L1), and to responses to test stimulus after conditioning and pre-conditioning stimuli (L2). Below, delays between (last) conditioning stimulus and test stimulus. B, latencies L0 and L1 as in A, and the latency (from the time of application of the test stimulus) to the response to the single conditioning impulse C1. C, latencies L0 and L2 as in A, and the latencies (from the test stimulus) to the responses to the conditioning impulses C2 and the pre-conditioning stimulus P2. NB, in A, the pre-conditioning impulse appears to accelerate the test impulse at short intervals but this is due to the effect of the pre-conditioning impulse in accelerating the conditioning impulse. The latency difference between time of arrival of the conditioning impulse and test impulse is always greater for two conditioning impulses (L2 − C2 in C) than for one (L1 − C1 in B).

Figure 6. Relationship between supernormality and activity-dependent slowing

Supernormality was estimated continuously by alternating single and double pulses, with interstimulus interval of 50 ms. Mean baseline stimulation rate 0.25 Hz. Open bars indicate mean stimulation rate of 2 Hz. A and B, two mechanically responsive units recorded at the same time. Upper, L0 is latency unconditioned response; L1 is latency after single conditioning stimulus at interval of 50 ms; C1 is latency (relative to test stimulus) of response to conditioning stimulus. The interval between C1 and L1 never approaches the entrainment interval (≈10 ms), so supernormality was not limited by entrainment. Lower, latency change, calculated as (L1 − L0)/L0 × 100, where L0 at the time of the conditioned response was estimated by linear interpolation. C, relationship between percentage latency change (i.e. subnormality or supernormality), as in A (1) and B (2), and slowing of unconditioned test impulse. At the start of the train there is a linear relationship between supernormality and activity-dependent slowing, but this breaks down, especially for unit 2, for which supernormality reaches a maximum of ≈4 %.

Figure 4. Recording effects of different stimulation rates on recovery cycles of CMR and CMI units

Modified raster plot of CMR (shorter latency) and CMI (longer latency) units, showing effects of 3 min pause in baseline stimulation at 0.25 Hz, 2 Hz tetanus, and recording of recovery cycles to single conditioning stimuli (open circles) at mean stimulation rates of 0.5, 1 and 2 Hz. The CMI was identified by the slowing at 0.25 Hz after the pause, and by the slower recovery after 2 Hz stimulation. The six corrected recovery cycles are plotted in Fig. 5A. Delay is the interval between conditioning and test stimuli. NB, starting at 19 min and in between recovery cycle recordings, a conditioning stimulus was delivered 50 ms before the test stimulus. This shows the graded transition from subnormality to supernormality as the fibres hyperpolarised (cf. Fig. 6).

Figure 5. Recovery cycle dependence on stimulation rate and fibre function

A, examples of corrected recovery cycles for four different types of C fibre recorded at 0.5, 1 and 2 Hz. The CMR and CMI units are those illustrated in Fig. 4. B, superimposed corrected recovery cycles for all units recorded at 1 Hz of the four types illustrated in A. The greater effect of stimulation rate on the recovery cycles of CMR and CMI than on cold and sympathetic units is related to the greater effect of stimulation rate on the unconditioned conduction velocity and membrane potential of the nociceptor fibres.

Figure 3. Recovery cycle correction, allowing for changes in interspike intervals during conduction along the nerve

A, uncorrected recovery cycles, expressed as percentage increase in latency. Circles calculated from data points in Fig. 2 as (L1 − L0)/L0 × 100 (thick-walled circles) and (L2 − L0)/L0 × 100 (thin-walled circles). Lines were calculated from corrected recovery cycles below by simulation. B, corrected recovery cycles, with instantaneous change in velocity expressed as a function of interspike interval. (For correction method see text.) Thick lines, single conditioning impulse; thin lines, two conditioning impulses. C, simulation of relative latency changes along the length of the axon, for interstimulus interval of 8 ms (arrowed in A), to show how pre-conditioning stimulus can appear to accelerate test impulse. Latencies expressed relative to baseline latency of first impulse, assumed to conduct at constant velocity. Circles, latencies inferred from measurements (see text for explanation of x, y and z). Lines, latencies calculated from corrected recovery cycles in B by integration of slowing over distance. Upper, single conditioning impulse causes only slight relative slowing of test impulse at interval of 8 ms. Lower, pre-conditioning impulse accelerates second conditioning impulse, starting 20 ms later, towards the entrainment interval of ≈9 ms. This in turn accelerates the test impulse as interspike interval increases above its initial value of 8 ms.

Figure 7. A second conditioning stimulus never increases supernormality

Upper traces, corrected recovery cycles for single (thick lines) and double (thin lines) conditioning stimuli. Each trace is mean of indicated number of units. Lower traces, increase in slowing due to pre-conditioning stimulus; thick lines, mean; thin lines, mean ± s.d. Groups of units selected with differing degrees of supernormality. A and B, CMR units exhibit only subnormality at 0.5 Hz, and small supernormality at 1 Hz, but effect of pre-conditioning stimulus is always a small increase in slowing. C, pre-conditioning impulse induces characteristic peak in extra slowing of cold fibres at 50–60 ms. D, pronounced and long-lasting supernormality of sympathetic fibres is not increased by pre-conditioning impulse.

Figure 8. Duration of refractoriness depends on fibre function

A, partial recovery cycles plotted with logarithmic interspike interval axis to show early parts more clearly. B, measurements of ‘refractory period’ (until velocity recovered to within 2.5 % of value at 50 ms) compared for four types of C fibre. Horizontal lines indicate geometric means, asterisks indicate statistical significance of differences between means (*P < 0.05, **P < 0.01, ***P < 0.001, unpaired t-test of difference in mean logarithmic refractory periods).

Figure 9. Time constants of recovery from supernormality or subnormality

A, approximately exponential components of nociceptor recovery cycles obtained by subtracting recovery cycle at 1 Hz from recovery cycle of same unit at 0.5 Hz: left, data for 13 CMR units superimposed; centre, same data averaged and fitted with an exponential decay curve; right, similar data for four CMI units measured at 1 Hz and 0.5 Hz. B, time constants fitted to mean recovery cycles of CMI, cold and sympathetic units which exhibited supernormality at 1 Hz. C, time constants for individual units of different types compared. CMR and CMI unit time constants were not significantly different, but recovery from superexcitability of cold units was faster and of sympathetic units was slower, especially at 0.5 Hz. Three lines to the right connect time constants of same unit tested at different stimulation rates.