Our results show that relatively short-wavelength (1.34 μm) radiant laser pulses of identical energy directed to the dorsum and the palm of the hand elicit qualitatively and quantitatively similar pain sensations, and evoke brain potentials of similar latency and amplitude. This finding indicates that first pain to heat does exist in glabrous skin, and suggests that similar nociceptive afferents mediate psychophysical and electrophysiological responses to thermal stimulation of glabrous and hairy skin in humans.
In addition, contact heat stimuli applied to the dorsum and the palm of the hand elicit pain sensations and evoke brain potentials as well, although with significantly lower signal-to-noise ratio compared to laser-evoked potentials. However, the palm has to be stimulated with a significantly higher thermode temperature than the dorsum in order to obtain similar pain sensations, and despite this higher temperature of stimulation brain responses have significantly longer latencies and smaller amplitudes. These findings are consistent with the thickness-dependent delay and attenuation of the temperature waveform at nociceptor level when conductive heating is applied. We suggest that the previously reported lack of first pain and microneurographical II-AMH responses following glabrous skin stimulation has been the result of a search bias consequent to the use of conductive heating and long-wavelength radiant heating (i.e. CO2 laser) as stimulation procedures.
Contact versus radiant heat nociceptive stimulation of hairy skin
Noxious heat stimuli are frequently used to study the nociceptive system, because they activate a nociceptive-specific transduction mechanism (Julius & Basbaum, 2001). Nociceptive nerve endings can be heated by either thermal conduction or thermal radiation. The advent of devices able to raise quickly the skin temperature by either infrared thermal radiation (laser stimulators) or thermal conduction (contact thermodes) allowed the recording of stimulus-evoked brain potentials (LEPs and CHEPs) in the electroencephalogram (Carmon et al. 1978; Chen et al. 2001). Whereas the possibility of recording CHEPs has been reported only recently (Chen et al. 2001), LEPs have been extensively used in basic and clinical research to study the temporal dynamics of nociceptive processing, and to date are the best available tool for assessing the function of nociceptive pathways in patients (Cruccu et al. 2004).
Our results show that, matching intensity of perception and stimulated territory, LEPs have a significantly higher signal-to-noise ratio than CHEPs (Figs 1–3 and 6). This finding is consistent with the different biophysical properties of radiant versus conductive heat–skin interactions. Compared to conductive heating, the infrared radiation produced by modern solid-state lasers (such as thulium-YAG or neodymium-YAP) has the following advantages, which are relevant for the interpretation of the results reported in the present study.
(1) Radiant heat activates the nociceptive afferents in a selective fashion (Plaghki & Mouraux, 2003). In contrast, contact thermodes unavoidably produce concomitant stimulation of low-threshold mechanoreceptors, which modulate the spinal transmission of both nociceptive and heat information (Nathan et al. 1986).
(2) Because the laser energy is confined to a narrow beam of nearly parallel monochromatic electromagnetic waves (i.e. the energy fluence is extremely high), the rise of surface skin temperature is particularly fast (≫ 1000°C s−1, compared to 70°C s−1 of fastest contact thermodes, Chen et al. 2001).
(4) As a consequence of (3), the temperature increase induced at the specific receptor depth by infrared radiation is reproducible between trials (although not necessarily reproducible between subjects when relatively short wavelengths are used, because of differences in skin reflectance). In contrast, because contact heat stimulators receive a feedback temperature signal from a thermocouple embedded in the surface of the stimulator, the temperature waveform at the nociceptor level is both delayed and attenuated by thermal conduction between the skin surface and nociceptive nerve terminals (Magerl & Treede, 1996). Notably, these time and intensity differences between temperature profiles at surface and nociceptor level become more pronounced as the heating rate increases (Tillman et al. 1995). Thus, the faster the rate of conducting heating applied, the more difficult the prediction of temperature at nociceptor level.
(5) When lasers with sufficiently high extinction lengths are applied, the temperature waveform at nociceptor level is relatively independent of thickness of the epidermis; in other words, the epidermis is transparent to this kind of radiant heat.
Following hairy skin stimulation, we observed that CHEPs had significantly longer latencies and significantly smaller amplitudes than LEPs (Figs 2–5 and 8, and Table 1).
The average latency of the first negative CHEP peak at the vertex (N2) was 159 ms longer than the corresponding LEP peak (Figs 4B and 9 and Table 1). The heat ramp of the thermode lasted 214 ms (i.e. the time spent in order to reach the target temperature (51°C) from the baseline temperature (36°C) at a rate of 70°C s−1). Because II-AMHs have a median thermal threshold of ∼46°C (Treede et al. 1995; 1998), their activation is expected to happen not earlier than the second half of this 214 ms- long ramp. In addition, because of the reasons outlined above, the temperature rise at nociceptor depth is expected to be delayed by thermal conduction from the skin surface. Besides the direct effect of rise time, a further contribution to the longer latency of the CHEP response may be ascribed to the weaker spatial and temporal summation of the nociceptive input (synchronization effect). Because of the physiological variance in nociceptor thresholds, the delayed temperature profile induced by contact heat stimuli excites nociceptors with slightly higher thresholds with a longer delay, making the afferent volley less synchronized and exerting a less effective spatial summation at central synapses.
The average amplitude of the first negative CHEP peak at the vertex (N2) was 10.1 μV smaller than the corresponding LEP peak (Fig. 9 and Table 1). This finding is certainly the result of at least two phenomenona. First, a smaller synchronization effect (i.e. weaker spatial and temporal summation of the afferent volley at the central synapses) when conductive heating is applied; this is consistent with the previously reported reduction of the amplitude of the brain and psychophysical responses when heat stimuli of the same energy but longer duration are applied (Pertovaara et al. 1988; Treede et al. 1994; Iannetti et al. 2004). Second, a more important nociceptor fatigue and habituation during contact heat stimulation; this is consistent with a recent report of significantly smaller subjective ratings and CHEP amplitudes when contact heat stimuli are applied to a fixed surface (i.e. as in the present study) as compared to when their position was varied after each trial (Greffrath et al. 2006).
It is worth highlighting that all the results described here are obtained from an automatic and unbiased analysis of single-trial N2 and P2 latency and amplitude values (Mayhew et al. 2006). This novel approach allowed us to compare fairly the amplitude values of LEP and CHEP peaks of single trials, without incurring the bias that is normally introduced by the latency jitter when EP amplitude values are measured in standard, time-locked averages. As the latency jitter of nociceptive-related response is significant because of the relatively slow conduction velocity of nociceptive fibres (Purves & Boyd, 1993), the signal averaged across trials is blurred, and its amplitude is lower than the average of amplitudes of single-trial responses (mostly because the latency jitter between trials causes the averaging of signals which are out of phase, Iannetti et al. 2005). Accordingly, in the present study, the average amplitude of single-trial EPs was bigger than the amplitude of standard averages (e.g. after hairy skin stimulation: LEPs: N2standard, 16 μV; N2single-trial, 24 μV; CHEPs: N2standard, 8 μV; N2single-trial, 14 μV). Given that CHEPs showed a higher latency jitter than LEPs both within and between subjects (Figs 4 and 6–8), if standard averaged data was used, overestimation of the reduction of amplitude observed in CHEPs (e.g. N2 after hairy skin stimulation: standard amplitudes, 16 versus 8 μV; single-trial amplitudes, 24 versus 14 μV) would occur. Independently of the modality of heating, these findings clearly indicate the need for a single-trial approach in the analysis of heat-evoked potentials, especially when meaningful and unbiased comparisons between conditions with different latencies and signal-to-noise ratios are required (e.g. experimental modulations of response amplitudes, and lesions of the nociceptive pathways in clinical practice).
Evidence for II-AMHs in glabrous skin
There is little doubt that II-AMHs are the peripheral afferents responsible for first pain sensation to heat in hairy skin, and that they conduct the volley that elicits LEPs in the central nervous system. II-AMHs are the only heat nociceptors with short response latency and graded response to heat (Treede et al. 1998), and their threshold distribution matches the threshold distribution of pricking pain and late LEPs (Treede et al. 1994).
Microneurographical recordings in monkeys failed to detect II-AMH responses to CO2 laser heat stimuli applied to glabrous skin (Treede et al. 1995) and first pain sensation is not evoked when CO2 laser heat stimuli are applied to the palm of the hand in humans (Campbell & LaMotte, 1983). Taken together these findings have led to the notion that first pain and II-AMHs are lacking in primate glabrous skin (Meyer et al. 2006).
Our results challenge this notion, because they show that Nd:YAP laser stimuli of identical energy applied to the dorsum and the palm of the hand elicit qualitatively and quantitatively indistinguishable sensations (Fig. 1), and elicit brain potentials with extremely similar latency and amplitude values (Fig. 3 and Table 1). It is worth noting that variations in baseline temperature can lead to misinterpretations when radiant heat stimuli are applied to the skin (Tjolsen et al. 1988), because the differential temperature increase induced by radiant heating is independent of the baseline skin temperature (i.e. the stimulus-induced temperature increase adds to the baseline, see Fig. 2 in Iannetti et al. 2004). We excluded this potential confounding factor from our results, by ensuring that baseline hairy and glabrous skin temperatures were similar at the beginning and at the end of the recording session (difference always < 1°C).
These findings provide strong evidence for the existence, in human glabrous skin, of a population of heat nociceptors with physiological features (activation threshold, response latency, spatial distribution and conduction velocity) very similar to II-AMHs, and indicate that these afferents mediate first pain to heat.
The notion of the lack of first pain to heat in glabrous skin is puzzling, when examined from a finalistic perspective. The ability to appreciate and react appropriately to the contact between the palm of the hand and hot objects seems especially important for survival, particularly in animals with an extreme ability for manipulation like primates. Indeed, a well-timed withdrawal reflex when the palm of the hand gets in contact with a hot saucepan when cooking is a common experience of everyday life. Our results provide experimental evidence for the neural circuitry subserving this common behaviour, and indicate that first pain to heat in glabrous skin does exist.
We believe that previous negative results could have been due to a search bias explained by the biophysics of heat skin interactions. Both microneurographical and psychophysical experiments that have failed to reveal fast responses to heat when glabrous skin was stimulated have been conducted using a CO2 laser controlled by radiometric feedback, with a temperature rise time of approximately 100 ms (Meyer et al. 1976). CO2 laser has a wavelength in the far infrared (10.6 μm). Because of the characteristics of reflectance, transmissions and absorption of the human epidermis, at this wavelength the extinction length is in the order of single micrometers (Hardy & Muschenheim, 1934; Plaghki & Mouraux, 2003) (i.e. radiation is nearly extinct well above the depth where nociceptive free nerve endings terminate; 20–570 μm below skin surface in hairy skin (Tillman et al. 1995)). For these reasons, the heating induced by a CO2 infrared radiation behaves rather similarly to the heating induced by a very fast contact heat stimulus (although the heat ramp of the CO2 stimulus can be several orders of magnitude greater than that of a contact thermode): once the CO2 radiation is absorbed in the very first micrometers of the epidermis, the heating is subsequently transmitted by thermal conduction to the deeper epidermal layers where the activation of nociceptors occurs. During this transmission, the temperature waveform is progressively delayed and attenuated in a depth-dependent process, as has been shown in simulation and modelling studies (Bromm & Treede, 1984; Spiegel et al. 2000; Plaghki & Mouraux, 2003). As a consequence, when CO2 radiant heat stimuli of an intensity effectively eliciting first pain in hairy skin are applied to skin territories where a thicker epidermal layer is interposed between nociceptors and the skin surface (such as the glabrous skin of the palm, where the thickness of the stratum corneum is at least twice that in hairy skin, Whitton & Everall, 1973; Nouveau-Richard et al. 2004; Mountcastle, 2005), they can easily fail to reach the nociceptor activation threshold. Alternatively, the threshold is reached but without an optimal heat ramp profile, especially when pulses with relatively long rise times are applied (e.g. 100 ms in Treede et al. 1995; between 200 and 250 ms in Campbell & LaMotte, 1983). We believe that these mechanisms could have constituted a bias in the search for the neural afferents mediating first pain sensations to heat in glabrous skin. This hypothesis is strengthened by several observations. First, contact heat stimuli of identical intensity elicit lower pain ratings when applied to glabrous skin, and only when the nominal thermode temperature is significantly increased similar pain ratings (although qualitatively described as ‘less pricking’ than hairy skin stimulation) are obtained (Fig. 1). Second, when glabrous skin is stimulated, CHEPs show a slower latency with a bigger jitter, and a smaller amplitude than CHEPs following hairy skin stimulation (Figs 3–5 and 9). Reasons for these findings are the longer delay and the higher variability in nociceptor activation time when the temperature profile is delayed and attenuated by the transmission through the thicker stratum corneum of the hand palm. Third, short-lasting CO2 laser stimuli at the intensity sufficient to elicit LEPs following hand dorsum stimulation are not able to elicit LEPs following palm stimulation (G. Cruccu, personal communication), and only when the intensity of stimulation is significantly increased, LEPs to glabrous skin stimulation appear (Towell et al. 1996).
Although only dedicated microneurographical recordings using short-wavelength laser pulses as test stimuli will permit a full characterization of the response properties of nociceptors mediating first pain and late LEPs following glabrous skin stimulation, our results provide strong evidence that a population of nociceptors with properties very similar to II-AMHs is responsible for the sensation of first pain to heat in glabrous skin in humans.