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Mechanothermal nociceptors in the scaly skin of the chicken leg
PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 3 1 8 - 9
Neuroscience Vol. 106, No. 3, pp. 643^652, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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MECHANOTHERMAL NOCICEPTORS IN THE SCALY SKIN OF THE CHICKEN LEG M. J. GENTLE,* V. TILSTON and D. E. F. MCKEEGAN Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS Scotland, UK
AbstractöElectrophysiological recordings were made from single sensory mechanothermal nociceptive a¡erent ¢bres in dissected nerve ¢laments of the para¢bular nerve innervating the scaly skin of the lower leg of the chicken. Two classes of mechanothermal nociceptors were identi¢ed consisting of 34 C ¢bres (conduction velocities 0.45^1.5 m/s, mean 1.08) and nine A-delta ¢bres (3^15 m/s, mean 6.34). The C ¢bre a¡erents had receptive ¢elds which were circular or elliptical in shape and ranged in size from 1 mm in diameter to 4U3 mm. Thresholds to mechanical stimulation in the C ¢bre a¡erents ranged from 0.3 to 33 g (median 1.5 g) and thermal thresholds were in the range 39^61³C (median 49.4³C). Stimulus^response curves to thermal and/or mechanical stimulation were recorded from 28 C ¢bre a¡erents and subjected to a linear regression analysis to determine whether they were best ¢tted by a linear, log or power function. The results were variable and no single function provided the best ¢t for all the responses. Of the ¢bres tested with both stimulus modalities (n = 17), only 12 ¢bres showed the same best ¢t for both stimuli; in the others the best ¢t regression lines di¡ered between stimuli. The response of the A-delta ¢bres to mechanical and thermal stimulation was very similar to the C ¢bres but the small number of A-delta ¢bres precluded any detailed statistical analysis. Comparison of the physiological properties of the C ¢bres in the leg with those previously identi¢ed in the beak showed that those in the leg had signi¢cantly lower thermal thresholds, but higher mechanical thresholds. The possible functional signi¢cance of these di¡erences is discussed. These ¢ndings are also discussed in a comparative context to identify similarities and di¡erences between mechanothermal nociceptors in birds and other vertebrates, relating these to their possible evolutionary and functional signi¢cance. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: avian, mechanothermal nociceptor, para¢bular nerve, scaly skin, skin a¡erents.
Nociceptors innervated with C ¢bres in hairy skin are readily sensitised to heat whereas those in glabrous skin do not have this property (Campbell and Meyer, 1983; LaMotte et al., 1983). Di¡erences have also been reported following mechanical stimulation. Meyer et al. (1991) reported that mechanical thresholds of C and A ¢bre nociceptors were similar in hairy skin but both groups of nociceptors had signi¢cantly higher thresholds in glabrous skin. Di¡erent properties of nociceptors in di¡erent areas of the body suggest functional specialisation. Bharali and Lisney (1992) demonstrated that not all C ¢bre nociceptors were capable of inducing plasma extravasation following antidromic stimulation with functional subgroups of nociceptor a¡erents present. Regional di¡erences in the ability of antidromic stimulation to induce neurogenic in£ammation have also been reported in the chicken (Gentle and Hunter, 1990). Antidromic stimulation of the trigeminal nerve gave rise to plasma extravasation in the skin of the side of the head and wattle, whereas stimulation of the para¢bular nerve, which innervates the skin of the lower leg and ankle (Gentle, 1992), did not produce plasma extravasation. Anatomically, the epidermis of the beak and legs of the chicken are similar, with both the rhamphotheca of the beak and the thick scales of the lower leg consisting of a thickened keratinised outer layer (Lucas and Stettenheim, 1972). Apart from a role in the maintenance
Cutaneous mechanothermal or polymodal nociceptors have been investigated in both the hairy and glabrous skin in a variety of mammals (Campbell and Meyer, 1996) but they have received much less systematic study in non-mammalian vertebrates. In birds, nociceptors have been described in the beak of the chicken (Roumy and Leitner, 1973; Breward, 1983; Gentle, 1989, 1991) and goose (Gottschaldt, 1985), in the feathered skin of the pigeon (Necker and Reiner, 1980) and in both the embryonic and newly hatched domestic chick (Koltzenburg and Lewin, 1997). There is evidence that nociceptors have di¡erent properties in di¡erent areas of the skin. Two types of A ¢bre mechanothermal nociceptors have been identi¢ed in the monkey (Treede et al., 1995), type I which is found in hairy and glabrous skin, and type II which occurs only in hairy skin. Type I receptors have relatively high thermal thresholds and sensitise to burn injury, whereas type II have lower thresholds and desensitise to burn injury.
*Corresponding author. Tel. : +44-131-527-4255; fax: +44-131-4400434. E-mail address: [email protected] (M. J. Gentle). Abbreviations : AMH, A-delta ¢bre mechanoheat a¡erent ; AMT, A-delta ¢bre mechanothermal a¡erent; CMT, C ¢bre mechanothermal a¡erent; CV, conduction velocity; VR1, vanilloid receptor 1. 643
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of body integrity, the skin of the beak and lower leg ful¢l a number of di¡erent functions. The beak has an important sensory role in feeding, environmental manipulation and aggression, and the lower leg is involved in thermoregulation. By recording the neural activity in single a¡erent nociceptive ¢bres the present study was designed to investigate any di¡erences in the physiological responses shown by the mechanothermal nociceptors in the lower leg compared to those previously reported from the trigeminal nerve innervating the beak (Gentle, 1989, 1991).
EXPERIMENTAL PROCEDURES
Animals and surgical preparation To minimise su¡ering, all experiments were performed under general anaesthesia with authorisation of a UK Home O¤ce project licence (60/1632) and with the approval of the Roslin Institute Ethical Committee. The experiments were performed on 20 adult Brown Leghorn hens (Gallus gallus domesticus) weighing approximately 1.5 kg each which were bred at Roslin Institute. The animals were initially anaesthetised with sodium pentobarbitone (Sagatal, Rhoªne Me¨rieux, Ireland) given i.v. (24^30 mg/kg body weight) which provided su¤cient duration of anaesthesia to cannulate the branchial vein, after which the birds were maintained under urethane (ethyl carbamate) anaesthesia (1.5 g/kg body weight) for the duration of the experiment. Heart rate was continuously monitored and body temperature maintained at 40³C by means of a heated blanket monitored with a rectal probe. The physiological properties of the nociceptors in the scaly skin of the lower leg between the ankle (artc. Intertarsalis) and the foot were investigated by recording the electrical activity from single a¡erent ¢bres dissected from the para¢bular nerve. The recording set up was similar to that used previously to record articular a¡erents in the ankle joint (Gentle, 1992, 1997; Gentle and Thorp, 1994). The bird was placed on the heating blanket with the left leg ¢xed lateral side uppermost. This was accomplished by screwing the femur to a metal plate attached to a rod, which was securely attached to the underlying table. The lower leg and foot were securely attached to a moulded plate by means of cyanoacrylate adhesive applied to the medial surface of the leg. The skin was incised laterally approximately half way along the femur. After partial removal of the overlying iliotibialis and ilio¢bularis muscles the para¢bular nerve was dissected free a few millimetres distal to the point where it leaves the main trunk of the tibial nerve. The para¢bular nerve was supported on a black Perspex platform and the nerve sheath removed. Small nerve ¢laments were dissected from the main nerve using sharpened Watchmakers' forceps. The recording site formed a deep depression that was ¢lled with liquid para¤n to prevent the nerve from drying.
using an isolated stimulator (DS2, Digitimer). The distance between stimulating and recording electrodes was approximately 100 mm and the CV was calculated from the time required for the evoked action potential to travel from the stimulating to the recording electrodes. Experimental design Following dissection of a nerve ¢lament the surface of the lower leg was probed with a hand-held probe (tip diameter 1 mm) to identify single slowly adapting mechanoreceptors. Mechanical thresholds were determined using calibrated nylon mono¢laments (von Frey hairs) ranging from 0.1 to 15 g. The CV of the ¢bre was established and the size and position of the receptive ¢eld determined using a probe. Thermal sensitivity was determined by heating the skin at a rate of 1³C/s up to 56³C using a prefocused quartz glass light bulb with built-in re£ector (A/231, 12 V, 100 W, Wotan) orientated vertical to the skin. The temperature was measured at the surface of the skin with a type K thermocouple placed in the centre of the bulb focus and the temperature was controlled by a feedback circuit. If the ¢bre responded before 56³C the unit was not subjected to any further increase in temperature above the threshold. The ¢rst temperature tested was determined by this initial threshold and to avoid sensitisation of the units the majority of the units were not stimulated above 56³C. Some units however had very high thresholds and in this case they were stimulated up to 62³C. A ramp and hold stimulus was used with the rate of temperature increase during the ramping set at 1³C/s and the hold 10 s in duration. The thermal stimulator was accurate to 0.5³C and the interval between stimuli was 2 min. Because of the wide range in thresholds the stimuli were presented in an ascending, non-random series. If the stimulus did not produce an increase in response from the previous stimulus no further stimulus was given. After thermal stimulation the unit was given a minimum of a 5 min period without stimulation followed by a series of ramp and hold mechanical stimuli. Quantitative mechanical stimuli were delivered using a 0.5 mm diameter tungsten probe mounted on a force transducer (Dynameter UF1, Ormed Engineering) attached to a feedback-controlled stepping-motor with a lead screw (Scat-01, Digitimer). The ramp time was set at 1 s and the total stimulus duration was 10 s. The stimulator had an accuracy of þ 1 g and a stimulus range of 2^100 g. In practice the stimulus characteristics were partially determined by the underlying tissue. For example, if the receptive ¢eld was situated on a large scale immediately over bone, there was little tissue elasticity and relatively few steps from the motor were required to achieve the selected force, producing a short ramp. If the receptive ¢eld was nearer the foot the scales tended to be smaller overlying more elastic tissue, which continued to deform during the stimulus period resulting in no extended plateau in stimulus intensity. Statistical probabilities were calculated using a Mann^ Whitney U-test. In all instances P 6 0.05 was considered statistically signi¢cant.
Electrophysiological recording
RESULTS
Dissected nerve ¢laments were lifted onto one pole of a bipolar silver wire electrode with the other pole connected to a small strand of the nerve sheath. The electrical activity was ampli¢ed using an a.c. preampli¢er (P15, Grass Instrument) with the signal displayed on a storage oscilloscope (5113, Tektronix) and stored on a FM tape recorder (Store4DS, Racal Recorders). Further analysis of individual units was performed using Spike II Analysis programme with a 1401 Interface (Cambridge Electronic Design). To determine conduction velocities (CV) the para¢bular nerve was dissected in the lower leg about 20 mm proximal to the ankle joint and placed over stimulating electrodes. The nerve was electrically stimulated with square-wave pulses ranging in duration from 0.2 ms for the faster ¢bres to 1 ms for the C ¢bres
A total of 43 single mechanothermal a¡erents were analysed in these experiments. These consisted of 34 C ¢bres (identi¢ed as units with conduction velocities less than 2 m/s) and nine A-delta ¢bres (conduction velocities 2^20 m/s). A large number of other a¡erent ¢bres were identi¢ed with properties similar to those previously reported in the chicken beak (Gentle, 1989). These units were not systematically investigated but on the basis of their response to stimulation with a mechanical probe and ice water were classi¢ed as rapidly adapting mechanoreceptors (Herbst and Grandry units), thermally
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Fig. 1. Histogram showing the frequency distribution of the conduction velocities of the nociceptor ¢bres recorded in the study.
and non-thermally sensitive slowly adapting mechanoreceptors, and cold receptors. C ¢bre mechanothermal a¡erents The CV of C ¢bre mechanothermal a¡erents (CMT) ranged from 0.45 to 2 m/s (mean 1.08, S.E.M. þ 0.08) (Fig. 1). Although a variety of di¡erent sized a¡erents were identi¢ed a large part of the sample consisted of ¢bres with CV in the 0.5^1 m/s range. The receptive ¢elds of the ¢bres were situated on the lateral surface of skin over the tarsometatarsus region of the leg (Fig. 2) between the ankle and the proximal end of the toes. Two thirds of the units (68%) had small spotlike receptive ¢elds 1^2 mm in diameter, with some having slightly elongated receptive ¢elds (3U1 mm). The other units (32%) had larger ¢elds up to 4U3 mm in size. The position of the receptive ¢eld in relation to the scales was variable. Some units with small ¢elds
were situated in the softer tissue between the scales and this was also the case in some units with larger ¢elds, which could only be stimulated between scales. Other units however responded to stimulation on a single scale or over a number of scales. None of the ¢bres showed any spontaneous activity. Thresholds to mechanical stimulation ranged from 0.1 to 33 g (median 1.5); the position and size of the mechanical thresholds are shown in Figs. 2 and 3. Comparison of the mechanical thresholds of units found in the skin adjacent to the ankle with those in the lower tarsometatarsus showed that signi¢cantly lower values were found over the ankle region (Fig. 2) (median: ankle, 1.2; lower tarsometatarsus, 3.1; P = 0.0046). For thermal thresholds (Fig. 4), which ranged from 39 to 61³C (median 49.4³C), there were no regional di¡erences in the lower leg. There was no correlation between mechanical and thermal thresholds for individual units. Of 28 ¢bres, 23 were tested with thermal stimulation,
Fig. 2. Photograph of a lateral view of the tarsometatarsus showing the position of the receptive ¢elds of the A-delta and C ¢bre nociceptors as well as the mechanical thresholds of the C ¢bres. D, A-delta ¢bres; a, less than 1 g CMT; b, 1^2 g CMT; O, 2^3 g CMT; R, 3^4 g CMT; E, more than 4 g CMT.
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Fig. 3. Histogram showing the thresholds of C ¢bre mechanothermal nociceptors to von Frey mechanical stimulation of the skin over the tarsometatarsus.
22 with mechanical stimulation and 17 with both stimulus modalities. Stimulus^response curves were constructed by plotting the number of impulses recorded from the stimulus onset to the end of the stimulus for the mechanical force applied and for the 10 s of the hold period for the thermal stimulus (Fig. 5). Because of the longer ramp time of the thermal stimulus, higher temperatures resulted in longer stimulus durations. Using only the 10 s hold period allowed comparison of stimulus^ response curves for both thermal and mechanical stimulation and also allowed the thermal response to be compared to previous ¢ndings (Beck et al., 1974; Breward, 1983, 1985; Gentle, 1989). There was considerable variability in the response characteristics of the units in terms of response magnitudes, shape and slope of the curves and the range over which they responded. An example of one of the ¢bres is shown in Fig. 6. In general, mechanical stimulation produced an increasing response to increasing stimulus intensity up to maximum after which any further increase in stimulus intensity resulted in a decline in response. Several of the units
responded to thermal stimulation in a similar manner. To reduce possible sensitisation few of the ¢bres were tested above 56³C, and as a result many displayed an increasing response up to the highest temperature tested. Maximum discharge rates varied between ¢bres and ranged from 1 to 14 impulses/s following mechanical stimulation (mean 7.2, S.E.M. þ 0.86, n = 22) and 3 to 18 impulses/s after thermal stimulation (mean 7.1, S.E.M. þ 0.9, n = 23). Some ¢bres showed the highest discharge rate to mechanical stimulation whereas others responded maximally to thermal stimulation. During mechanical stimulation, 90% of the ¢bres exhibited the highest discharge rate during the ramp or at the top of the ramp. The most rapid discharge rate was during the hold phase of the stimulus in only two ¢bres. Higher levels of mechanical stimulation resulted in higher discharge rates during the ramp phase of the stimulus and a lower irregular discharge during the hold phase (Fig. 6). Thermal stimulation produced a slightly di¡erent response with half of the ¢bres showing their highest discharge rate at the top of the ramp and half during
Fig. 4. Histogram showing the thresholds of C ¢bre mechanothermal nociceptors to thermal stimulation of the skin of the tarsometatarsus.
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Fig. 5. Stimulus^response curves of the A-delta and C ¢bre mechanothermal nociceptors in response to a ramp and hold mechanical or thermal stimulation of the skin of the tarsometatarsus: (a) A-delta ¢bres mechanical stimulation; (b) C ¢bres mechanical stimulation ; (c) A-delta ¢bres thermal stimulation; (d) C ¢bres thermal stimulation.
the hold. In general the ¢bres showed an irregular rate of discharge to thermal stimulation and this was also seen at low levels of mechanical stimulation. The data were subjected to regression analysis to get a more quantitative measure of the stimulus^response relationship. This analysis was performed on the untransformed data (linear function), semi-log transformation (relating the response to the log of the stimulus, log function) and a log^log transformation (power function). Because some units reached a peak in response the regression lines were calculated on the threshold to peak values or the maximum response recorded. The percentage of units where the r2 values were highest is shown in Table 1. Following mechanical stimulation 41% were best ¢tted by a linear function whereas the power function best ¢tted in 66% of units following thermal stimulation. In 12 of the 17 units tested with both mechanical and thermal stimulation the stimulus^ response function was the same for both stimulus modalities. In the other units the best ¢t regression lines differed between the stimuli. A-delta ¢bre mechanothermal a¡erents A-delta ¢bre mechanothermal a¡erent (AMT) units were found much less frequently than the CMT units
and therefore these results are based on only nine ¢bres, limiting the scope for statistical comparisons. The CV of the AMT units ranged from 3 to 15 m/s (mean 6.34, S.E.M. þ 1.27). The receptive ¢elds of the ¢bres (Fig. 2), like those of the CMT, were found over the entire lateral surface of the tarsometatarsus. Four ¢bres had small spot-like receptive ¢elds 1^2 mm in diameter with the other ¢ve having many ¢elds up to 7U3 mm, much larger than those identi¢ed in the CMT ¢bres. The position of the receptive ¢elds in relation to the scales was variable. Thresholds to mechanical stimulation were between 0.3 and 5.5 g (median 1.2 g) and thermal thresholds ranged from 45 to 56³C (median 49³C). These values are similar to those found in the CMT ¢bres, and like the CMT units, none of the AMT ¢bres showed any spontaneous activity. Stimulus^response curves were recorded for eight ¢bres of which eight were tested with mechanical stimulation and six with both thermal and mechanical stimulation. An example of one of the ¢bres is shown in Fig. 6. There was considerable variability in the response characteristics of the AMT ¢bres which fell within the ranges shown by CMT ¢bres. Maximum discharge rates varied between ¢bres and ranged from 4 to 33 impulses/s (mean 13.6, S.E.M. þ 4.2) for mechanical stimulation and
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Fig. 6. Example of an A-delta (AMT) and a C (CMT) ¢bre mechanothermal nociceptor responding to both mechanical and thermal stimulation. The upper trace is the stimulus transducer output and the lower trace the ¢bre discharge. Both ¢bres show an irregular discharge to suprathreshold thermal stimulation with an increase in response with increasing stimulus intensity. Mechanical stimulation resulted in an irregular discharge at low stimulus intensities, a high rate of discharge during the ramp and the early part of the hold phase followed by a decline in response before the end of the stimulus.
3 to 13 impulses/s (mean 5.8, S.E.M. þ 1.6) with thermal stimulation. Like the CMT ¢bres, highest discharge rates were generally seen during the ramp phase of the stimulus during mechanical stimulation. Following thermal
stimulation four units responded maximally during the hold phase and two during the ramp. The same regression analysis was applied to the AMT ¢bres, and unlike the CMT ¢bres none of the AMT
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Table 1. The percentage of A-delta and C ¢bre nociceptors that were best ¢tted by either a linear, logarithmic or power intensity function determined by a linear regression analysis of their stimulus^response curves to mechanical and thermal stimulation
A-delta nociceptor Mechanical stimulus (n = 7) A-delta nociceptor Thermal stimulus (n = 6) C nociceptor Mechanical stimulus (n = 22) C nociceptor Thermal stimulus (n = 23)
% r2 % r2 % r2 % r2
units ( þ S.E.M.) units ( þ S.E.M.) units ( þ S.E.M.) units ( þ S.E.M.)
Linear
Log
Power
0
43 0.94 (0.015) 33 0.858 (0.013) 23 0.928 (0.027) 17 0.914 (0.034)
57 0.903 67 0.947 36 0.926 66 0.923
0 41 0.862 (0.076) 17 0.966 (0.004)
(0.034) (0.018) (0.026) (0.027)
2
Mean correlation coe¤cients r þ S.E.M.
¢bres best ¢tted a linear function (Table 1). Of the six units tested with both mechanical and thermal stimulation, four had stimulus^response functions which were the same for both stimulus modalities. Comparison of physiological properties of the CMT receptors found in the leg with those in the beak A limited physiological comparison can be made between the receptors recorded from the scaly skin of the leg and those previously recorded in our laboratory from the beak (Breward, 1985; Gentle, 1989). Beak receptors had signi¢cantly lower thermal thresholds than those found in the tarsometatarsus (beak median 46³C; tarsometatarsal median 50³C; P = 0.007) but higher mechanical thresholds (beak median 16.5 g; tarsometatarsal median 1.5 g; P = 0.001). The receptive ¢elds of beak receptors tended to be circular or elliptical in shape with 1^2 mm diameters (Gentle, 1989, 1991). While 68% of the tarsometatarsal receptive ¢elds investigated in the current study were similar in size and shape, other leg receptors had receptive ¢elds which were considerably larger (up to 4U3 mm). Detailed stimulus^response characteristics have only been described for beak CMT receptors following thermal stimulation. In general, the stimulus^response curves for the beak receptors were very similar to those found in the leg. Following regression analysis, the best ¢tting functions for beak receptors were 64% power, 19% linear, and 12% log, with 5% of units showing a poor correlation with all three functions. These values do not di¡er from those found in the tarsometatarsus (Table 1).
DISCUSSION
Both C and A-delta ¢bre mechanothermal nociceptors were found in the scaly skin on the tarsometatarsus of the chicken. AMT ¢bres were not found in previous studies of the chicken beak (Breward, 1983, 1985; Gentle, 1989, 1991) but their presence cannot be completely ruled out because the CV was not measured for all of the ¢bres reported and some AMT ¢bres could have been mistaken for CMTs. The presence of AMT ¢bres in the scaly skin of the leg but not in the beak is of interest because the similar type II A-delta ¢bre mechanoheat a¡erent (AMH) receptors found in mam-
mals also show regional di¡erences in distribution. They are found only in hairy skin of man and primates, for example (Meyer and Campbell, 1981; Campbell and LaMotte, 1983; Torebjo«rk et al., 1996). AMT ¢bres form a substantial population of heatresponsive neurones in the chick embryo (Koltzenburg and Lewin, 1997), with 36% of the A ¢bres responding to noxious heat. However, none did so in the hatchling chick, leading Koltzenburg and Lewin to conclude that the A ¢bres lost their heat sensitivity after hatching. The presence of AMT ¢bres in the adult bird contradicts these conclusions and would suggest that either the sample size in the chick study (16 neurones) was too small, or that heat-responsive properties are re-acquired later in development. There have been few detailed studies of nociceptors in other avian species. Both A-delta and C ¢bre nociceptors have been reported in the feather skin of the pigeon (Necker and Reiner, 1980), although in this experiment the CV were not measured and the ¢bre type was assumed from the size and shape of the action potentials. The scaly skin of the chicken tarsometatarsus is structurally very similar to the reptilian (Rundall, 1947; Spearman, 1971). Rapidly adapting and slowly adapting mechanoreceptors have been reported in reptilian skin (Simino¡, 1968; Simino¡ and Kruger, 1968; Proske, 1969) but nociceptors have not been systematically studied. Similar receptors were observed in the present study and have also been described more fully in the chicken beak (Gentle, 1989). Two possible nociceptors were described in the alligator (Kenton et al., 1971) but not enough details were provided to allow a comparison with the avian receptors reported here. The AMT receptors in the chicken skin show a slowly adapting response with an orderly relationship between stimulus intensity and response rate to both mechanical and thermal stimulation. These properties are similar to type II AMH receptors in mammals (Meyer et al., 1985; Treede et al., 1995, 1998). A-delta ¢bre nociceptors are found less frequently than C ¢bre nociceptors in mammalian skin nerves and this was also the case in the chicken. There is recent evidence to suggest that the reduced frequency of type II AMH receptors is due to search techniques which rely on mechanical responsiveness at low thresholds (Burgess and Perl, 1967; Georgopoulos, 1976; Fitzgerald and Lynn, 1977), since type II receptors have signi¢cantly
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higher mechanical thresholds than type I AMH ¢bres (Treede et al., 1998). Thermal thresholds of the chicken AMT units (median 49³C) were slightly higher than those found in the human (37^47³C) or monkey (median 45³C) skin (Torebjo«rk et al., 1996; Campbell and Meyer, 1996) but this di¡erence is not unexpected given that temperature was measured at the skin surface and the thick keratinised scales will have insulating properties. The mechanical response properties of mammalian AMH ¢bres have received much less attention than their thermal responses. One complicating factor is the relationship between the size of the probe delivering the stimulus and the force applied. Thus a given pressure did not evoke the same response in cat AMH nociceptors as the probe size was varied (Garell et al., 1996). In response to a ramp and hold stimulus the AMT nociceptors found in the chicken leg were similar to the AMH nociceptors found in the feline hairy skin (Garell et al., 1996) in terms of threshold and pattern of response. The stimulus^response curves for thermal and mechanical stimulation in chicken skin AMT and CMT ¢bres were also similar. One of the prominent features which distinguish the mechanical response properties of A and C ¢bre nociceptors in mammals is that at a given force the A ¢bres exhibit a much higher rate of activity (Handwerker et al., 1987; Garell et al., 1996). This was not seen in the relatively few units recorded in the chicken skin. The thermal thresholds of the CMT nociceptors in the tarsometatarsal skin of the chicken were within the ranges described in various mammalian species (Bessou and Perl, 1969; Beck et al., 1974; Beitel and Dubner, 1976; Georgopoulos, 1977; Kumazawa and Perl, 1977; Lynn, 1979; Lynn and Carpenter, 1982; LaMotte et al., 1982; Fleischer et al., 1983) and were signi¢cantly higher than those found in the beak (Gentle, 1989). Both structures are covered with a thick outer keratinised layer and the functional signi¢cance of these thermal threshold differences is not immediately apparent. The lower leg has an important role in thermoregulation and is possibly subjected to more extremes of temperature than the beak. The similarities in the thermal properties of the AMT and CMT nociceptors in the bird and mammal are of interest in relation to thermal transduction mechanisms. It has been found that in mammals the product of the vanilloid receptor 1 (VR1) gene has the characteristics of the low threshold heat transducer (Caterina et al., 1997; Nagy and Rang, 1999). In cultured dorsal root ganglion neurones both the rat and chick cells showed a similar response to temperature and while all low threshold heat-sensitive rat cells responded to capsaicin none of the chick neurones responded to it (Nagy and Rang, 2000). Although the chick neurones did not respond to capsaicin the response to low threshold noxious heat stimuli was antagonised by the competitive capsaicin antagonist capsazepine (Marin-Burgin et al., 2000). The absence of response to capsaicin in the chick indicates that the chick VR1 receptor is not identical to that found
in the rat but the response to capsazepine suggests that it may be a homologue of the VR1 receptor. In general, mechanical thresholds to von Frey stimulation were similar in the chicken to those found in mammals (Lynn, 1994; Garell et al., 1996). The signi¢cantly lower mechanical thresholds seen at the ankle compared to the lower tarsometatarsus were similar to the reducing von Frey thresholds from the leg to the digits found in the rat hind limb (Lynn and Carpenter, 1982). Beak CMT ¢bres had signi¢cantly higher mechanical thresholds compared to those found in the leg and the functional signi¢cance of this may lie in the high forces sustained by the beak when pecking. Anyone who has experienced being pecked by a chicken can testify to the pain in£icted. Di¡erences in mechanical thresholds in di¡erent skin areas have been reported in other species. For example, CMH thresholds in the hairy skin of the monkey were signi¢cantly lower than in glabrous skin (2.2 and 5.6 bar respectively, Treede et al., 1995). It should be noted, however, that these di¡erences are smaller than the 10-fold di¡erences observed in the chicken. Following suprathreshold mechanical or thermal stimulation the CMT receptors showed a slowly adapting response with a greater response with increasing stimulus intensity. Stimulus^response curves to both thermal and mechanical stimulation have not been previously systematically investigated, and relatively few studies have attempted to ¢t intensity functions to stimulus^response curves. Using thermal responses from the rabbit and cat, Lynn (1979) and Fleischer et al. (1983) found individual units ¢tting a log response function (Lynn, 1979) or pooled data ¢tting a power function (Fleischer et al., 1983). In a study of primate trigeminal nociceptors, Beitel and Dubner (1976) found that out of eight C ¢bre nociceptors ¢ve were best ¢tted by a power function, two by a linear and one by a log function. These ¢ndings are very similar to the nociceptors in the chicken tarsometatarsus and beak (Breward, 1985) in which 66% power, 17% log, and 17% linear functions were best ¢tted. The situation was slightly di¡erent following mechanical stimulation of the tarsometatarsal skin, where a greater number of units were ¢tted by a linear function. In the chicken, 70% of units showed the same stimulus^response function to each stimulus modality. About half of the units reported here responded with maximum discharge rates to the thermal rather than mechanical stimulation; in the others mechanical stimulation produced the greatest response. The pattern of response of the CMT units to a ramp and hold suprathreshold stimulus di¡ered depending on the stimulus modality. Thermal stimulation resulted in an irregular discharge and the pattern of response varied between units. Responses to mechanical stimulation were less variable, with the majority of ¢bres showing the highest rates of discharge during the ramp phase followed by a decline during the hold. A similar pattern of discharge has been observed in rat (White and Levine, 1991) and cat (Garell et al., 1996) C ¢bre nociceptors.
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Chicken leg mechanothermal nociceptors CONCLUSION
These experiments demonstrate that the scaly skin of the chicken leg contains nociceptors which can detect and transmit information relating to the position and intensity of potentially noxious mechanical and thermal stimulation. The physiological properties of these nociceptors resemble the type II AMH and CMH nociceptors found in mammals that have similar properties over a wide variety of species and body locations. This raises two interesting questions. Firstly, what is the evolutionary signi¢cance of these ¢ndings and secondly, what is the role of the AMT receptors in the pain perceived by birds. Our current state of knowledge makes it di¤cult to say at what stage in evolution vertebrates acquired mechanothermal nociceptors. Birds and mammals are not closely related in evolutionary terms, and it seems likely that similar nociceptors were present in their reptilian common ancestors. There have been no detailed studies on reptilian nociceptors and the data from lower vertebrates are fragmentary. Nociceptors have been reported in amphibians (review by Spray, 1976; Stevens, 1992) but there is insu¤cient detail to compare them to avian or mammalian nociceptors. There is electrophysiological evidence for nociceptors in cyclostomes (Martin and Wickelgren, 1971; Matthews and Wickelgren, 1978) but in elasmobranches there is a virtual lack of unmyelinated sensory a¡erents (Snow et al., 1993) and Leonard (1985) could produce no evidence for
651
nociceptors. In teleosts there have been no studies but there is some recent evidence that both C and A-delta cutaneous a¡erents are present in the trigeminal system of the trout (Sneddon and Gentle, unpublished observations). The presence of AMT ¢bres has implications for the possible pain experience by the animal and there is growing evidence that birds experience pain rather than showing simple nociceptive responses (Gentle and Hill, 1987; Gentle et al., 1991, 1997; Gentle and Corr, 1995; Gentle and Tilston, 1999). In man, heat stimuli evoke a double pain sensation with the ¢rst sensation a sharp pricking sensation and the second sensation a burning feeling (Lewis and Pochin, 1937; Campbell and LaMotte, 1983). The latency to respond to ¢rst pain is too quick to be carried by slowly conducting C ¢bres and the type II AMH ¢bres are thought to signal ¢rst pain. The presence of similar ¢bres in the chicken raises the possibility of double pain but it is di¤cult to conceive of an experiment which could accurately test for it. It is likely that in the chicken leg, A-delta nociceptors are responsible for re£ex leg withdrawal which has an obvious protective function, maintaining the integrity of the leg and foot by responding rapidly to noxious stimulation.
AcknowledgementsöThis work was partly funded by the UK Ministry of Agriculture, Fisheries and Food.
REFERENCES
Beck, P.W., Handwerker, H.O., Zimmermann, M., 1974. Nervous out£ow from the cat's foot during noxious radiant heat stimulation. Brain Res. 67, 373^386. Beitel, R.E., Dubner, R., 1976. Responses of unmyelinated (C) polymodal nociceptors to thermal stimuli applied to monkey's face. J. Neurophysiol. 39, 1160^1175. Bessou, P., Perl, E.R., 1969. Response of cutaneous sensory units with unmyelinated ¢bres to noxious stimuli. J. Neurophysiol. 32, 1025^1043. Bharali, L.A.M., Lisney, S.J.W., 1992. The relationship between unmyelinated a¡erent type and neurogenic plasma extravasation in normal and reinnervated rat skin. Neuroscience 47, 703^712. Breward, J., 1983. Cutaneous nociceptors in the chicken beak. J. Physiol. 346, 56P. Breward, J., 1985. An electrophysiological investigation of the e¡ects of beak trimming in the domestic fowl (Gallus gallus domesticus). Ph.D. thesis, University of Edinburgh. Burgess, P.R., Perl, E.R., 1967. Myelinated a¡erent ¢bres responding speci¢cally to noxious stimulation of the skin. J. Physiol. 190, 541^562. Campbell, J.N., LaMotte, R.H., 1983. Latency to detection of ¢rst pain. Brain Res. 266, 203^208. Campbell, J.N., Meyer, R.A., 1996. Cutaneous nociceptors. In: Belmonte, C., Cervero, F. (Eds.), Neurobiology of Nociceptors. Oxford University Press, Oxford, pp. 117^145. Campbell, J.N., Meyer, R.A., 1983. Sensitization of unmyelinated nociceptive a¡erents in the monkey varies with skin type. J. Neurophysiol. 49, 98^110. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: heat-activated ion channel in the pain pathway. Nature 389, 816^824. Fitzgerald, M., Lynn, B., 1977. The sensitization of high threshold mechanoreceptors with myelinated axons by repeated heating. J. Physiol. 265, 549^563. Fleischer, E., Handwerker, H.O., Joukhadar, S., 1983. Unmyelinated nociceptive units in two skin areas of the rat. Brain Res. 267, 81^92. Garell, P.C., McGillis, S.L.B., Greenspan, J.D., 1996. Mechanical response properties of nociceptors innervating feline hairy skin. J. Neurophysiol. 75, 1177^1189. Gentle, M.J., 1989. Cutaneous sensory a¡erents recorded from the nervus intramandibularis of Gallus gallus var domesticus. J. Comp. Physiol. A 164, 763^774. Gentle, M.J., 1991. The acute e¡ects of amputation on peripheral trigeminal a¡erents in Gallus gallus var domesticus. Pain 46, 97^103. Gentle, M.J., 1992. Ankle joint (artc. intertarsalis) receptors in domestic fowl. Neuroscience 49, 991^1000. Gentle, M.J., 1997. Sodium urate arthritis : e¡ects on the sensory properties of articular a¡erents in the chicken. Pain 70, 245^251. Gentle, M.J., Corr, S.A., 1995. Endogenous analgesia in the chicken. Neurosci. Lett. 201, 211^214. Gentle, M.J., Hill, F.L., 1987. Oral lesions in the chicken: behavioural responses following nociceptive stimulation. Physiol. Behav. 40, 781^783. Gentle, M.J., Hunter, L.N., 1990. Physiological and behavioural responses associated with feather removal in Gallus gallus var domesticus. Res. Vet. Sci. 50, 95^101. Gentle, M.J., Thorp, B.H., 1994. Sensory properties of ankle joint capsule mechanoreceptors in acute monoarthritic chickens. Pain 57, 61^374. Gentle, M.J., Tilston, V.L., 1999. Reduction in peripheral in£ammation by changes in attention. Physiol. Behav. 66, 289^292.
NSC 5159 26-9-01
652
M. J. Gentle et al.
Gentle, M.J., Hunter, L.N., Corr, S.A., 1997. E¡ects of caudolateral neostriatal ablations on pain-related behaviour in the chicken. Physiol. Behav. 61, 493^498. Gentle, M.J., Hunter, L.N., Waddington, D., 1991. The onset of pain related behaviours following partial beak amputation in the chicken. Neurosci. Lett. 128, 113^116. Georgopoulos, A.P., 1976. Functional properties of primary a¡erent units probably related to pain mechanisms in primate glabrous skin. J. Neurophysiol. 39, 71^83. Georgopoulos, A.P., 1977. Stimulus-response relations in high-threshold mechanothermal ¢bers innervating primate glabrous skin. Brain Res. 128, 547^552. Gottschaldt, K.M., 1985. Structure and function of avian somatosensory receptors. In: King, A.S., McLelland, J. (Eds.), Form and Function in Birds, Vol. 3. Academic Press, London, pp. 375^461. Handwerker, H.O., Anton, F., Reeh, P.W., 1987. Discharge patterns of a¡erent cutaneous nerve ¢bers from the rat's tail during prolonged noxious mechanical stimulation. Exp. Brain Res. I 65, 493^504. Kenton, B., Kruger, L., Woo, M., 1971. Two classes of slowly adapting mechanoreceptor ¢bres in reptile cutaneous nerve. J. Physiol. 212, 21^44. Koltzenburg, M., Lewin, G.R., 1997. Receptive properties of embryonic chick sensory neurons innervating skin. J. Neurophysiol. 78, 2560^2568. Kumazawa, T., Perl, E.R., 1977. Primate cutaneous sensory units with unmyelinated (C) ¢bers. J. Neurophysiol. 40, 1325^1338. LaMotte, R.H., Thalhammer, J.G., Robinson, C.J., 1983. Peripheral neural correlates of magnitude of cutaneous pain and hyperalgesia: a comparison of neural events in monkey with sensory judgements in humans. J. Neurophysiol. 50, 1^26. LaMotte, R.H., Thalhammer, J.G., Torebjo«rk, H.E., Robinson, C.J., 1982. Peripheral neural mechanisms of cutaneous hyperalgesia following mild injury by heat. J. Neurosci. 2, 765^781. Leonard, R.B., 1985. Primary a¡erent receptive ¢eld properties and neurotransmitter candidates in a vertebrate lacking unmyelinated ¢bres. Prog. Clin. Biol. Res. 176, 135^145. Lewis, T., Pochin, E.E., 1937. The double pain response of the human skin to a single stimulus. Clin. Sci. 3, 67^76. Lucas, A.M., Stettenheim, P.R., 1972. Avian Anatomy. Integument Part II. United States Department of Agriculture, Washington. Lynn, B., 1979. The heat sensitization of polymodal nociceptors in the rabbit and its independence of local blood supply. J. Physiol. 287, 493^507. Lynn, B., 1994. The ¢bre composition of cutaneous nerves and the classi¢cation and response properties of cutaneous a¡erents, with particular reference to nociception. Pain Rev. 1, 172^183. Lynn, B., Carpenter, S.E., 1982. Primary a¡erent units from the hairy skin of the rat hind limb. Brain Res. 238, 29^43. Marin-Burgin, A., Reppenhagen, S., Klusch, A., Wendland, J.R., Petersen, M., 2000. Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitive. Eur. J. Neurosci. 12, 3560^3566. Martin, A.R., Wickelgren, W.O., 1971. Sensory cells in the spinal cord of the sea lamprey. J. Physiol. 212, 65^83. Matthews, G., Wickelgren, W.O., 1978. Trigeminal sensory neurons of sea lamprey. J. Comp. Physiol. 123, 329^333. Meyer, R.A., Campbell, J.N., 1981. Myelinated nociceptive a¡erents account for the hyperalgesia that follows a burn to the hand. Science 213, 1527^1529. Meyer, R.A., Campbell, J.N., Raja, S.N., 1985. Peripheral neural mechanisms of cutaneous hyperalgesia. In: Fields, H.L., Dubner, R., Cervero, F. (Eds.), Advances in Pain Research and Therapy. Raven Press, New York, pp. 53^71. Meyer, R.A., Davis, K.D., Cohen, R.H., Treede, R.D., Campbell, J.N., 1991. Mechanically insensitive a¡erents (MIAs) in cutaneous nerves of monkey. Brain Res. 561, 252^261. Nagy, I., Rang, H., 1999. Noxious heat activates all capsaicin-sensitive and also a sub-population of capsaicin-insensitive dorsal root ganglion neurons. Neuroscience 88, 995^997. Nagy, I., Rang, H., 2000. Comparison of currents activated by noxious heat in rat and chicken primary sensory neurons. Reg. Peptides 96, 3^6. Necker, R., Reiner, B., 1980. Temperature-sensitive mechanoreceptors, thermoreceptors and heat nociceptors in the feathered skin of pigeons. J. Comp. Physiol. A 135, 201^207. Proske, U., 1969. Vibration-sensitive mechanoreceptors in snake skin. Exp. Neurol. 23, 187^194. Roumy, M., Leitner, L.M., 1973. Activites a¡erentes provenant de bec superieur de la poule domestique. C.R. Acad. Sci. Paris 277, 1791^1794. Rundall, K.M., 1947. X-ray studies of the distribution of protein chain types in the vertebrate epidermis. Biochim. Biophys. Acta 1, 549^563. Snow, P.J., Plenderleith, M.B., Wright, L.L., 1993. Quantitative study of primary sensory neurone populations of three species of elasmobranch ¢sh. J. Comp. Neurol. 334, 97^103. Spray, D.C., 1976. Pain and temperature receptors of anurans. In: Llinas, R., Precht, W. (Eds.), Frog Neurobiology. Springer Verlag, Berlin, pp. 607^628. Spearman, R.I.C., 1971. Integumentary system. In: Bell, D.J., Freeman, B.M. (Eds.), Physiology and Biochemistry of the Domestic Fowl. Academic Press, London, pp. 603^620. Stevens, C.W., 1992. Alternatives to the use of mammals for pain research. Life Sci. 50, 901^912. Simino¡, R., 1968. Quantitative properties of slowly adapting mechanoreceptors in alligator skin. Exp. Neurol. 21, 290^306. Simino¡, R., Kruger, L., 1968. Properties of reptilian cutaneous mechanoreceptors. Exp. Neurol. 20, 403^414. Torebjo«rk, H.E., Schmelz, M., Handwerker, H.O., 1996. Functional properties of human cutaneous nociceptors and their role in pain and hyperalgesia. In: Belmonte, C., Cervero, F. (Eds.), Neurobiology of Nociceptors. Oxford University Press, Oxford, pp. 349^369. Treede, R.D., Meyer, R.A., Campbell, J.N., 1998. Myelinated mechanically insensitive a¡erents from monkey hairy skin: heat-response properties. J. Neurophysiol. 80, 1082^1093. Treede, R.D., Meyer, R.A., Raja, S.N., Campbell, J.N., 1995. Evidence for two di¡erent heat transduction mechanisms in nociceptive primary a¡erents innervating monkey skin. J. Physiol. 483, 747^758. White, D.M., Levine, J.D., 1991. Di¡erent mechanical transduction mechanisms for the immediate and delayed responses of rat C-¢ber nociceptors. J. Neurophysiol. 66, 363^368. (Accepted 21 July 2001)
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Neuroscience Vol. 106, No. 3, pp. 643^652, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
www.elsevier.com/locate/neuroscience
MECHANOTHERMAL NOCICEPTORS IN THE SCALY SKIN OF THE CHICKEN LEG M. J. GENTLE,* V. TILSTON and D. E. F. MCKEEGAN Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS Scotland, UK
AbstractöElectrophysiological recordings were made from single sensory mechanothermal nociceptive a¡erent ¢bres in dissected nerve ¢laments of the para¢bular nerve innervating the scaly skin of the lower leg of the chicken. Two classes of mechanothermal nociceptors were identi¢ed consisting of 34 C ¢bres (conduction velocities 0.45^1.5 m/s, mean 1.08) and nine A-delta ¢bres (3^15 m/s, mean 6.34). The C ¢bre a¡erents had receptive ¢elds which were circular or elliptical in shape and ranged in size from 1 mm in diameter to 4U3 mm. Thresholds to mechanical stimulation in the C ¢bre a¡erents ranged from 0.3 to 33 g (median 1.5 g) and thermal thresholds were in the range 39^61³C (median 49.4³C). Stimulus^response curves to thermal and/or mechanical stimulation were recorded from 28 C ¢bre a¡erents and subjected to a linear regression analysis to determine whether they were best ¢tted by a linear, log or power function. The results were variable and no single function provided the best ¢t for all the responses. Of the ¢bres tested with both stimulus modalities (n = 17), only 12 ¢bres showed the same best ¢t for both stimuli; in the others the best ¢t regression lines di¡ered between stimuli. The response of the A-delta ¢bres to mechanical and thermal stimulation was very similar to the C ¢bres but the small number of A-delta ¢bres precluded any detailed statistical analysis. Comparison of the physiological properties of the C ¢bres in the leg with those previously identi¢ed in the beak showed that those in the leg had signi¢cantly lower thermal thresholds, but higher mechanical thresholds. The possible functional signi¢cance of these di¡erences is discussed. These ¢ndings are also discussed in a comparative context to identify similarities and di¡erences between mechanothermal nociceptors in birds and other vertebrates, relating these to their possible evolutionary and functional signi¢cance. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: avian, mechanothermal nociceptor, para¢bular nerve, scaly skin, skin a¡erents.
Nociceptors innervated with C ¢bres in hairy skin are readily sensitised to heat whereas those in glabrous skin do not have this property (Campbell and Meyer, 1983; LaMotte et al., 1983). Di¡erences have also been reported following mechanical stimulation. Meyer et al. (1991) reported that mechanical thresholds of C and A ¢bre nociceptors were similar in hairy skin but both groups of nociceptors had signi¢cantly higher thresholds in glabrous skin. Di¡erent properties of nociceptors in di¡erent areas of the body suggest functional specialisation. Bharali and Lisney (1992) demonstrated that not all C ¢bre nociceptors were capable of inducing plasma extravasation following antidromic stimulation with functional subgroups of nociceptor a¡erents present. Regional di¡erences in the ability of antidromic stimulation to induce neurogenic in£ammation have also been reported in the chicken (Gentle and Hunter, 1990). Antidromic stimulation of the trigeminal nerve gave rise to plasma extravasation in the skin of the side of the head and wattle, whereas stimulation of the para¢bular nerve, which innervates the skin of the lower leg and ankle (Gentle, 1992), did not produce plasma extravasation. Anatomically, the epidermis of the beak and legs of the chicken are similar, with both the rhamphotheca of the beak and the thick scales of the lower leg consisting of a thickened keratinised outer layer (Lucas and Stettenheim, 1972). Apart from a role in the maintenance
Cutaneous mechanothermal or polymodal nociceptors have been investigated in both the hairy and glabrous skin in a variety of mammals (Campbell and Meyer, 1996) but they have received much less systematic study in non-mammalian vertebrates. In birds, nociceptors have been described in the beak of the chicken (Roumy and Leitner, 1973; Breward, 1983; Gentle, 1989, 1991) and goose (Gottschaldt, 1985), in the feathered skin of the pigeon (Necker and Reiner, 1980) and in both the embryonic and newly hatched domestic chick (Koltzenburg and Lewin, 1997). There is evidence that nociceptors have di¡erent properties in di¡erent areas of the skin. Two types of A ¢bre mechanothermal nociceptors have been identi¢ed in the monkey (Treede et al., 1995), type I which is found in hairy and glabrous skin, and type II which occurs only in hairy skin. Type I receptors have relatively high thermal thresholds and sensitise to burn injury, whereas type II have lower thresholds and desensitise to burn injury.
*Corresponding author. Tel. : +44-131-527-4255; fax: +44-131-4400434. E-mail address: [email protected] (M. J. Gentle). Abbreviations : AMH, A-delta ¢bre mechanoheat a¡erent ; AMT, A-delta ¢bre mechanothermal a¡erent; CMT, C ¢bre mechanothermal a¡erent; CV, conduction velocity; VR1, vanilloid receptor 1. 643
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of body integrity, the skin of the beak and lower leg ful¢l a number of di¡erent functions. The beak has an important sensory role in feeding, environmental manipulation and aggression, and the lower leg is involved in thermoregulation. By recording the neural activity in single a¡erent nociceptive ¢bres the present study was designed to investigate any di¡erences in the physiological responses shown by the mechanothermal nociceptors in the lower leg compared to those previously reported from the trigeminal nerve innervating the beak (Gentle, 1989, 1991).
EXPERIMENTAL PROCEDURES
Animals and surgical preparation To minimise su¡ering, all experiments were performed under general anaesthesia with authorisation of a UK Home O¤ce project licence (60/1632) and with the approval of the Roslin Institute Ethical Committee. The experiments were performed on 20 adult Brown Leghorn hens (Gallus gallus domesticus) weighing approximately 1.5 kg each which were bred at Roslin Institute. The animals were initially anaesthetised with sodium pentobarbitone (Sagatal, Rhoªne Me¨rieux, Ireland) given i.v. (24^30 mg/kg body weight) which provided su¤cient duration of anaesthesia to cannulate the branchial vein, after which the birds were maintained under urethane (ethyl carbamate) anaesthesia (1.5 g/kg body weight) for the duration of the experiment. Heart rate was continuously monitored and body temperature maintained at 40³C by means of a heated blanket monitored with a rectal probe. The physiological properties of the nociceptors in the scaly skin of the lower leg between the ankle (artc. Intertarsalis) and the foot were investigated by recording the electrical activity from single a¡erent ¢bres dissected from the para¢bular nerve. The recording set up was similar to that used previously to record articular a¡erents in the ankle joint (Gentle, 1992, 1997; Gentle and Thorp, 1994). The bird was placed on the heating blanket with the left leg ¢xed lateral side uppermost. This was accomplished by screwing the femur to a metal plate attached to a rod, which was securely attached to the underlying table. The lower leg and foot were securely attached to a moulded plate by means of cyanoacrylate adhesive applied to the medial surface of the leg. The skin was incised laterally approximately half way along the femur. After partial removal of the overlying iliotibialis and ilio¢bularis muscles the para¢bular nerve was dissected free a few millimetres distal to the point where it leaves the main trunk of the tibial nerve. The para¢bular nerve was supported on a black Perspex platform and the nerve sheath removed. Small nerve ¢laments were dissected from the main nerve using sharpened Watchmakers' forceps. The recording site formed a deep depression that was ¢lled with liquid para¤n to prevent the nerve from drying.
using an isolated stimulator (DS2, Digitimer). The distance between stimulating and recording electrodes was approximately 100 mm and the CV was calculated from the time required for the evoked action potential to travel from the stimulating to the recording electrodes. Experimental design Following dissection of a nerve ¢lament the surface of the lower leg was probed with a hand-held probe (tip diameter 1 mm) to identify single slowly adapting mechanoreceptors. Mechanical thresholds were determined using calibrated nylon mono¢laments (von Frey hairs) ranging from 0.1 to 15 g. The CV of the ¢bre was established and the size and position of the receptive ¢eld determined using a probe. Thermal sensitivity was determined by heating the skin at a rate of 1³C/s up to 56³C using a prefocused quartz glass light bulb with built-in re£ector (A/231, 12 V, 100 W, Wotan) orientated vertical to the skin. The temperature was measured at the surface of the skin with a type K thermocouple placed in the centre of the bulb focus and the temperature was controlled by a feedback circuit. If the ¢bre responded before 56³C the unit was not subjected to any further increase in temperature above the threshold. The ¢rst temperature tested was determined by this initial threshold and to avoid sensitisation of the units the majority of the units were not stimulated above 56³C. Some units however had very high thresholds and in this case they were stimulated up to 62³C. A ramp and hold stimulus was used with the rate of temperature increase during the ramping set at 1³C/s and the hold 10 s in duration. The thermal stimulator was accurate to 0.5³C and the interval between stimuli was 2 min. Because of the wide range in thresholds the stimuli were presented in an ascending, non-random series. If the stimulus did not produce an increase in response from the previous stimulus no further stimulus was given. After thermal stimulation the unit was given a minimum of a 5 min period without stimulation followed by a series of ramp and hold mechanical stimuli. Quantitative mechanical stimuli were delivered using a 0.5 mm diameter tungsten probe mounted on a force transducer (Dynameter UF1, Ormed Engineering) attached to a feedback-controlled stepping-motor with a lead screw (Scat-01, Digitimer). The ramp time was set at 1 s and the total stimulus duration was 10 s. The stimulator had an accuracy of þ 1 g and a stimulus range of 2^100 g. In practice the stimulus characteristics were partially determined by the underlying tissue. For example, if the receptive ¢eld was situated on a large scale immediately over bone, there was little tissue elasticity and relatively few steps from the motor were required to achieve the selected force, producing a short ramp. If the receptive ¢eld was nearer the foot the scales tended to be smaller overlying more elastic tissue, which continued to deform during the stimulus period resulting in no extended plateau in stimulus intensity. Statistical probabilities were calculated using a Mann^ Whitney U-test. In all instances P 6 0.05 was considered statistically signi¢cant.
Electrophysiological recording
RESULTS
Dissected nerve ¢laments were lifted onto one pole of a bipolar silver wire electrode with the other pole connected to a small strand of the nerve sheath. The electrical activity was ampli¢ed using an a.c. preampli¢er (P15, Grass Instrument) with the signal displayed on a storage oscilloscope (5113, Tektronix) and stored on a FM tape recorder (Store4DS, Racal Recorders). Further analysis of individual units was performed using Spike II Analysis programme with a 1401 Interface (Cambridge Electronic Design). To determine conduction velocities (CV) the para¢bular nerve was dissected in the lower leg about 20 mm proximal to the ankle joint and placed over stimulating electrodes. The nerve was electrically stimulated with square-wave pulses ranging in duration from 0.2 ms for the faster ¢bres to 1 ms for the C ¢bres
A total of 43 single mechanothermal a¡erents were analysed in these experiments. These consisted of 34 C ¢bres (identi¢ed as units with conduction velocities less than 2 m/s) and nine A-delta ¢bres (conduction velocities 2^20 m/s). A large number of other a¡erent ¢bres were identi¢ed with properties similar to those previously reported in the chicken beak (Gentle, 1989). These units were not systematically investigated but on the basis of their response to stimulation with a mechanical probe and ice water were classi¢ed as rapidly adapting mechanoreceptors (Herbst and Grandry units), thermally
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Fig. 1. Histogram showing the frequency distribution of the conduction velocities of the nociceptor ¢bres recorded in the study.
and non-thermally sensitive slowly adapting mechanoreceptors, and cold receptors. C ¢bre mechanothermal a¡erents The CV of C ¢bre mechanothermal a¡erents (CMT) ranged from 0.45 to 2 m/s (mean 1.08, S.E.M. þ 0.08) (Fig. 1). Although a variety of di¡erent sized a¡erents were identi¢ed a large part of the sample consisted of ¢bres with CV in the 0.5^1 m/s range. The receptive ¢elds of the ¢bres were situated on the lateral surface of skin over the tarsometatarsus region of the leg (Fig. 2) between the ankle and the proximal end of the toes. Two thirds of the units (68%) had small spotlike receptive ¢elds 1^2 mm in diameter, with some having slightly elongated receptive ¢elds (3U1 mm). The other units (32%) had larger ¢elds up to 4U3 mm in size. The position of the receptive ¢eld in relation to the scales was variable. Some units with small ¢elds
were situated in the softer tissue between the scales and this was also the case in some units with larger ¢elds, which could only be stimulated between scales. Other units however responded to stimulation on a single scale or over a number of scales. None of the ¢bres showed any spontaneous activity. Thresholds to mechanical stimulation ranged from 0.1 to 33 g (median 1.5); the position and size of the mechanical thresholds are shown in Figs. 2 and 3. Comparison of the mechanical thresholds of units found in the skin adjacent to the ankle with those in the lower tarsometatarsus showed that signi¢cantly lower values were found over the ankle region (Fig. 2) (median: ankle, 1.2; lower tarsometatarsus, 3.1; P = 0.0046). For thermal thresholds (Fig. 4), which ranged from 39 to 61³C (median 49.4³C), there were no regional di¡erences in the lower leg. There was no correlation between mechanical and thermal thresholds for individual units. Of 28 ¢bres, 23 were tested with thermal stimulation,
Fig. 2. Photograph of a lateral view of the tarsometatarsus showing the position of the receptive ¢elds of the A-delta and C ¢bre nociceptors as well as the mechanical thresholds of the C ¢bres. D, A-delta ¢bres; a, less than 1 g CMT; b, 1^2 g CMT; O, 2^3 g CMT; R, 3^4 g CMT; E, more than 4 g CMT.
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Fig. 3. Histogram showing the thresholds of C ¢bre mechanothermal nociceptors to von Frey mechanical stimulation of the skin over the tarsometatarsus.
22 with mechanical stimulation and 17 with both stimulus modalities. Stimulus^response curves were constructed by plotting the number of impulses recorded from the stimulus onset to the end of the stimulus for the mechanical force applied and for the 10 s of the hold period for the thermal stimulus (Fig. 5). Because of the longer ramp time of the thermal stimulus, higher temperatures resulted in longer stimulus durations. Using only the 10 s hold period allowed comparison of stimulus^ response curves for both thermal and mechanical stimulation and also allowed the thermal response to be compared to previous ¢ndings (Beck et al., 1974; Breward, 1983, 1985; Gentle, 1989). There was considerable variability in the response characteristics of the units in terms of response magnitudes, shape and slope of the curves and the range over which they responded. An example of one of the ¢bres is shown in Fig. 6. In general, mechanical stimulation produced an increasing response to increasing stimulus intensity up to maximum after which any further increase in stimulus intensity resulted in a decline in response. Several of the units
responded to thermal stimulation in a similar manner. To reduce possible sensitisation few of the ¢bres were tested above 56³C, and as a result many displayed an increasing response up to the highest temperature tested. Maximum discharge rates varied between ¢bres and ranged from 1 to 14 impulses/s following mechanical stimulation (mean 7.2, S.E.M. þ 0.86, n = 22) and 3 to 18 impulses/s after thermal stimulation (mean 7.1, S.E.M. þ 0.9, n = 23). Some ¢bres showed the highest discharge rate to mechanical stimulation whereas others responded maximally to thermal stimulation. During mechanical stimulation, 90% of the ¢bres exhibited the highest discharge rate during the ramp or at the top of the ramp. The most rapid discharge rate was during the hold phase of the stimulus in only two ¢bres. Higher levels of mechanical stimulation resulted in higher discharge rates during the ramp phase of the stimulus and a lower irregular discharge during the hold phase (Fig. 6). Thermal stimulation produced a slightly di¡erent response with half of the ¢bres showing their highest discharge rate at the top of the ramp and half during
Fig. 4. Histogram showing the thresholds of C ¢bre mechanothermal nociceptors to thermal stimulation of the skin of the tarsometatarsus.
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Fig. 5. Stimulus^response curves of the A-delta and C ¢bre mechanothermal nociceptors in response to a ramp and hold mechanical or thermal stimulation of the skin of the tarsometatarsus: (a) A-delta ¢bres mechanical stimulation; (b) C ¢bres mechanical stimulation ; (c) A-delta ¢bres thermal stimulation; (d) C ¢bres thermal stimulation.
the hold. In general the ¢bres showed an irregular rate of discharge to thermal stimulation and this was also seen at low levels of mechanical stimulation. The data were subjected to regression analysis to get a more quantitative measure of the stimulus^response relationship. This analysis was performed on the untransformed data (linear function), semi-log transformation (relating the response to the log of the stimulus, log function) and a log^log transformation (power function). Because some units reached a peak in response the regression lines were calculated on the threshold to peak values or the maximum response recorded. The percentage of units where the r2 values were highest is shown in Table 1. Following mechanical stimulation 41% were best ¢tted by a linear function whereas the power function best ¢tted in 66% of units following thermal stimulation. In 12 of the 17 units tested with both mechanical and thermal stimulation the stimulus^ response function was the same for both stimulus modalities. In the other units the best ¢t regression lines differed between the stimuli. A-delta ¢bre mechanothermal a¡erents A-delta ¢bre mechanothermal a¡erent (AMT) units were found much less frequently than the CMT units
and therefore these results are based on only nine ¢bres, limiting the scope for statistical comparisons. The CV of the AMT units ranged from 3 to 15 m/s (mean 6.34, S.E.M. þ 1.27). The receptive ¢elds of the ¢bres (Fig. 2), like those of the CMT, were found over the entire lateral surface of the tarsometatarsus. Four ¢bres had small spot-like receptive ¢elds 1^2 mm in diameter with the other ¢ve having many ¢elds up to 7U3 mm, much larger than those identi¢ed in the CMT ¢bres. The position of the receptive ¢elds in relation to the scales was variable. Thresholds to mechanical stimulation were between 0.3 and 5.5 g (median 1.2 g) and thermal thresholds ranged from 45 to 56³C (median 49³C). These values are similar to those found in the CMT ¢bres, and like the CMT units, none of the AMT ¢bres showed any spontaneous activity. Stimulus^response curves were recorded for eight ¢bres of which eight were tested with mechanical stimulation and six with both thermal and mechanical stimulation. An example of one of the ¢bres is shown in Fig. 6. There was considerable variability in the response characteristics of the AMT ¢bres which fell within the ranges shown by CMT ¢bres. Maximum discharge rates varied between ¢bres and ranged from 4 to 33 impulses/s (mean 13.6, S.E.M. þ 4.2) for mechanical stimulation and
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Fig. 6. Example of an A-delta (AMT) and a C (CMT) ¢bre mechanothermal nociceptor responding to both mechanical and thermal stimulation. The upper trace is the stimulus transducer output and the lower trace the ¢bre discharge. Both ¢bres show an irregular discharge to suprathreshold thermal stimulation with an increase in response with increasing stimulus intensity. Mechanical stimulation resulted in an irregular discharge at low stimulus intensities, a high rate of discharge during the ramp and the early part of the hold phase followed by a decline in response before the end of the stimulus.
3 to 13 impulses/s (mean 5.8, S.E.M. þ 1.6) with thermal stimulation. Like the CMT ¢bres, highest discharge rates were generally seen during the ramp phase of the stimulus during mechanical stimulation. Following thermal
stimulation four units responded maximally during the hold phase and two during the ramp. The same regression analysis was applied to the AMT ¢bres, and unlike the CMT ¢bres none of the AMT
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Table 1. The percentage of A-delta and C ¢bre nociceptors that were best ¢tted by either a linear, logarithmic or power intensity function determined by a linear regression analysis of their stimulus^response curves to mechanical and thermal stimulation
A-delta nociceptor Mechanical stimulus (n = 7) A-delta nociceptor Thermal stimulus (n = 6) C nociceptor Mechanical stimulus (n = 22) C nociceptor Thermal stimulus (n = 23)
% r2 % r2 % r2 % r2
units ( þ S.E.M.) units ( þ S.E.M.) units ( þ S.E.M.) units ( þ S.E.M.)
Linear
Log
Power
0
43 0.94 (0.015) 33 0.858 (0.013) 23 0.928 (0.027) 17 0.914 (0.034)
57 0.903 67 0.947 36 0.926 66 0.923
0 41 0.862 (0.076) 17 0.966 (0.004)
(0.034) (0.018) (0.026) (0.027)
2
Mean correlation coe¤cients r þ S.E.M.
¢bres best ¢tted a linear function (Table 1). Of the six units tested with both mechanical and thermal stimulation, four had stimulus^response functions which were the same for both stimulus modalities. Comparison of physiological properties of the CMT receptors found in the leg with those in the beak A limited physiological comparison can be made between the receptors recorded from the scaly skin of the leg and those previously recorded in our laboratory from the beak (Breward, 1985; Gentle, 1989). Beak receptors had signi¢cantly lower thermal thresholds than those found in the tarsometatarsus (beak median 46³C; tarsometatarsal median 50³C; P = 0.007) but higher mechanical thresholds (beak median 16.5 g; tarsometatarsal median 1.5 g; P = 0.001). The receptive ¢elds of beak receptors tended to be circular or elliptical in shape with 1^2 mm diameters (Gentle, 1989, 1991). While 68% of the tarsometatarsal receptive ¢elds investigated in the current study were similar in size and shape, other leg receptors had receptive ¢elds which were considerably larger (up to 4U3 mm). Detailed stimulus^response characteristics have only been described for beak CMT receptors following thermal stimulation. In general, the stimulus^response curves for the beak receptors were very similar to those found in the leg. Following regression analysis, the best ¢tting functions for beak receptors were 64% power, 19% linear, and 12% log, with 5% of units showing a poor correlation with all three functions. These values do not di¡er from those found in the tarsometatarsus (Table 1).
DISCUSSION
Both C and A-delta ¢bre mechanothermal nociceptors were found in the scaly skin on the tarsometatarsus of the chicken. AMT ¢bres were not found in previous studies of the chicken beak (Breward, 1983, 1985; Gentle, 1989, 1991) but their presence cannot be completely ruled out because the CV was not measured for all of the ¢bres reported and some AMT ¢bres could have been mistaken for CMTs. The presence of AMT ¢bres in the scaly skin of the leg but not in the beak is of interest because the similar type II A-delta ¢bre mechanoheat a¡erent (AMH) receptors found in mam-
mals also show regional di¡erences in distribution. They are found only in hairy skin of man and primates, for example (Meyer and Campbell, 1981; Campbell and LaMotte, 1983; Torebjo«rk et al., 1996). AMT ¢bres form a substantial population of heatresponsive neurones in the chick embryo (Koltzenburg and Lewin, 1997), with 36% of the A ¢bres responding to noxious heat. However, none did so in the hatchling chick, leading Koltzenburg and Lewin to conclude that the A ¢bres lost their heat sensitivity after hatching. The presence of AMT ¢bres in the adult bird contradicts these conclusions and would suggest that either the sample size in the chick study (16 neurones) was too small, or that heat-responsive properties are re-acquired later in development. There have been few detailed studies of nociceptors in other avian species. Both A-delta and C ¢bre nociceptors have been reported in the feather skin of the pigeon (Necker and Reiner, 1980), although in this experiment the CV were not measured and the ¢bre type was assumed from the size and shape of the action potentials. The scaly skin of the chicken tarsometatarsus is structurally very similar to the reptilian (Rundall, 1947; Spearman, 1971). Rapidly adapting and slowly adapting mechanoreceptors have been reported in reptilian skin (Simino¡, 1968; Simino¡ and Kruger, 1968; Proske, 1969) but nociceptors have not been systematically studied. Similar receptors were observed in the present study and have also been described more fully in the chicken beak (Gentle, 1989). Two possible nociceptors were described in the alligator (Kenton et al., 1971) but not enough details were provided to allow a comparison with the avian receptors reported here. The AMT receptors in the chicken skin show a slowly adapting response with an orderly relationship between stimulus intensity and response rate to both mechanical and thermal stimulation. These properties are similar to type II AMH receptors in mammals (Meyer et al., 1985; Treede et al., 1995, 1998). A-delta ¢bre nociceptors are found less frequently than C ¢bre nociceptors in mammalian skin nerves and this was also the case in the chicken. There is recent evidence to suggest that the reduced frequency of type II AMH receptors is due to search techniques which rely on mechanical responsiveness at low thresholds (Burgess and Perl, 1967; Georgopoulos, 1976; Fitzgerald and Lynn, 1977), since type II receptors have signi¢cantly
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higher mechanical thresholds than type I AMH ¢bres (Treede et al., 1998). Thermal thresholds of the chicken AMT units (median 49³C) were slightly higher than those found in the human (37^47³C) or monkey (median 45³C) skin (Torebjo«rk et al., 1996; Campbell and Meyer, 1996) but this di¡erence is not unexpected given that temperature was measured at the skin surface and the thick keratinised scales will have insulating properties. The mechanical response properties of mammalian AMH ¢bres have received much less attention than their thermal responses. One complicating factor is the relationship between the size of the probe delivering the stimulus and the force applied. Thus a given pressure did not evoke the same response in cat AMH nociceptors as the probe size was varied (Garell et al., 1996). In response to a ramp and hold stimulus the AMT nociceptors found in the chicken leg were similar to the AMH nociceptors found in the feline hairy skin (Garell et al., 1996) in terms of threshold and pattern of response. The stimulus^response curves for thermal and mechanical stimulation in chicken skin AMT and CMT ¢bres were also similar. One of the prominent features which distinguish the mechanical response properties of A and C ¢bre nociceptors in mammals is that at a given force the A ¢bres exhibit a much higher rate of activity (Handwerker et al., 1987; Garell et al., 1996). This was not seen in the relatively few units recorded in the chicken skin. The thermal thresholds of the CMT nociceptors in the tarsometatarsal skin of the chicken were within the ranges described in various mammalian species (Bessou and Perl, 1969; Beck et al., 1974; Beitel and Dubner, 1976; Georgopoulos, 1977; Kumazawa and Perl, 1977; Lynn, 1979; Lynn and Carpenter, 1982; LaMotte et al., 1982; Fleischer et al., 1983) and were signi¢cantly higher than those found in the beak (Gentle, 1989). Both structures are covered with a thick outer keratinised layer and the functional signi¢cance of these thermal threshold differences is not immediately apparent. The lower leg has an important role in thermoregulation and is possibly subjected to more extremes of temperature than the beak. The similarities in the thermal properties of the AMT and CMT nociceptors in the bird and mammal are of interest in relation to thermal transduction mechanisms. It has been found that in mammals the product of the vanilloid receptor 1 (VR1) gene has the characteristics of the low threshold heat transducer (Caterina et al., 1997; Nagy and Rang, 1999). In cultured dorsal root ganglion neurones both the rat and chick cells showed a similar response to temperature and while all low threshold heat-sensitive rat cells responded to capsaicin none of the chick neurones responded to it (Nagy and Rang, 2000). Although the chick neurones did not respond to capsaicin the response to low threshold noxious heat stimuli was antagonised by the competitive capsaicin antagonist capsazepine (Marin-Burgin et al., 2000). The absence of response to capsaicin in the chick indicates that the chick VR1 receptor is not identical to that found
in the rat but the response to capsazepine suggests that it may be a homologue of the VR1 receptor. In general, mechanical thresholds to von Frey stimulation were similar in the chicken to those found in mammals (Lynn, 1994; Garell et al., 1996). The signi¢cantly lower mechanical thresholds seen at the ankle compared to the lower tarsometatarsus were similar to the reducing von Frey thresholds from the leg to the digits found in the rat hind limb (Lynn and Carpenter, 1982). Beak CMT ¢bres had signi¢cantly higher mechanical thresholds compared to those found in the leg and the functional signi¢cance of this may lie in the high forces sustained by the beak when pecking. Anyone who has experienced being pecked by a chicken can testify to the pain in£icted. Di¡erences in mechanical thresholds in di¡erent skin areas have been reported in other species. For example, CMH thresholds in the hairy skin of the monkey were signi¢cantly lower than in glabrous skin (2.2 and 5.6 bar respectively, Treede et al., 1995). It should be noted, however, that these di¡erences are smaller than the 10-fold di¡erences observed in the chicken. Following suprathreshold mechanical or thermal stimulation the CMT receptors showed a slowly adapting response with a greater response with increasing stimulus intensity. Stimulus^response curves to both thermal and mechanical stimulation have not been previously systematically investigated, and relatively few studies have attempted to ¢t intensity functions to stimulus^response curves. Using thermal responses from the rabbit and cat, Lynn (1979) and Fleischer et al. (1983) found individual units ¢tting a log response function (Lynn, 1979) or pooled data ¢tting a power function (Fleischer et al., 1983). In a study of primate trigeminal nociceptors, Beitel and Dubner (1976) found that out of eight C ¢bre nociceptors ¢ve were best ¢tted by a power function, two by a linear and one by a log function. These ¢ndings are very similar to the nociceptors in the chicken tarsometatarsus and beak (Breward, 1985) in which 66% power, 17% log, and 17% linear functions were best ¢tted. The situation was slightly di¡erent following mechanical stimulation of the tarsometatarsal skin, where a greater number of units were ¢tted by a linear function. In the chicken, 70% of units showed the same stimulus^response function to each stimulus modality. About half of the units reported here responded with maximum discharge rates to the thermal rather than mechanical stimulation; in the others mechanical stimulation produced the greatest response. The pattern of response of the CMT units to a ramp and hold suprathreshold stimulus di¡ered depending on the stimulus modality. Thermal stimulation resulted in an irregular discharge and the pattern of response varied between units. Responses to mechanical stimulation were less variable, with the majority of ¢bres showing the highest rates of discharge during the ramp phase followed by a decline during the hold. A similar pattern of discharge has been observed in rat (White and Levine, 1991) and cat (Garell et al., 1996) C ¢bre nociceptors.
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Chicken leg mechanothermal nociceptors CONCLUSION
These experiments demonstrate that the scaly skin of the chicken leg contains nociceptors which can detect and transmit information relating to the position and intensity of potentially noxious mechanical and thermal stimulation. The physiological properties of these nociceptors resemble the type II AMH and CMH nociceptors found in mammals that have similar properties over a wide variety of species and body locations. This raises two interesting questions. Firstly, what is the evolutionary signi¢cance of these ¢ndings and secondly, what is the role of the AMT receptors in the pain perceived by birds. Our current state of knowledge makes it di¤cult to say at what stage in evolution vertebrates acquired mechanothermal nociceptors. Birds and mammals are not closely related in evolutionary terms, and it seems likely that similar nociceptors were present in their reptilian common ancestors. There have been no detailed studies on reptilian nociceptors and the data from lower vertebrates are fragmentary. Nociceptors have been reported in amphibians (review by Spray, 1976; Stevens, 1992) but there is insu¤cient detail to compare them to avian or mammalian nociceptors. There is electrophysiological evidence for nociceptors in cyclostomes (Martin and Wickelgren, 1971; Matthews and Wickelgren, 1978) but in elasmobranches there is a virtual lack of unmyelinated sensory a¡erents (Snow et al., 1993) and Leonard (1985) could produce no evidence for
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nociceptors. In teleosts there have been no studies but there is some recent evidence that both C and A-delta cutaneous a¡erents are present in the trigeminal system of the trout (Sneddon and Gentle, unpublished observations). The presence of AMT ¢bres has implications for the possible pain experience by the animal and there is growing evidence that birds experience pain rather than showing simple nociceptive responses (Gentle and Hill, 1987; Gentle et al., 1991, 1997; Gentle and Corr, 1995; Gentle and Tilston, 1999). In man, heat stimuli evoke a double pain sensation with the ¢rst sensation a sharp pricking sensation and the second sensation a burning feeling (Lewis and Pochin, 1937; Campbell and LaMotte, 1983). The latency to respond to ¢rst pain is too quick to be carried by slowly conducting C ¢bres and the type II AMH ¢bres are thought to signal ¢rst pain. The presence of similar ¢bres in the chicken raises the possibility of double pain but it is di¤cult to conceive of an experiment which could accurately test for it. It is likely that in the chicken leg, A-delta nociceptors are responsible for re£ex leg withdrawal which has an obvious protective function, maintaining the integrity of the leg and foot by responding rapidly to noxious stimulation.
AcknowledgementsöThis work was partly funded by the UK Ministry of Agriculture, Fisheries and Food.
REFERENCES
Beck, P.W., Handwerker, H.O., Zimmermann, M., 1974. Nervous out£ow from the cat's foot during noxious radiant heat stimulation. Brain Res. 67, 373^386. Beitel, R.E., Dubner, R., 1976. Responses of unmyelinated (C) polymodal nociceptors to thermal stimuli applied to monkey's face. J. Neurophysiol. 39, 1160^1175. Bessou, P., Perl, E.R., 1969. Response of cutaneous sensory units with unmyelinated ¢bres to noxious stimuli. J. Neurophysiol. 32, 1025^1043. Bharali, L.A.M., Lisney, S.J.W., 1992. The relationship between unmyelinated a¡erent type and neurogenic plasma extravasation in normal and reinnervated rat skin. Neuroscience 47, 703^712. Breward, J., 1983. Cutaneous nociceptors in the chicken beak. J. Physiol. 346, 56P. Breward, J., 1985. An electrophysiological investigation of the e¡ects of beak trimming in the domestic fowl (Gallus gallus domesticus). Ph.D. thesis, University of Edinburgh. Burgess, P.R., Perl, E.R., 1967. Myelinated a¡erent ¢bres responding speci¢cally to noxious stimulation of the skin. J. Physiol. 190, 541^562. Campbell, J.N., LaMotte, R.H., 1983. Latency to detection of ¢rst pain. Brain Res. 266, 203^208. Campbell, J.N., Meyer, R.A., 1996. Cutaneous nociceptors. In: Belmonte, C., Cervero, F. (Eds.), Neurobiology of Nociceptors. Oxford University Press, Oxford, pp. 117^145. Campbell, J.N., Meyer, R.A., 1983. Sensitization of unmyelinated nociceptive a¡erents in the monkey varies with skin type. J. Neurophysiol. 49, 98^110. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: heat-activated ion channel in the pain pathway. Nature 389, 816^824. Fitzgerald, M., Lynn, B., 1977. The sensitization of high threshold mechanoreceptors with myelinated axons by repeated heating. J. Physiol. 265, 549^563. Fleischer, E., Handwerker, H.O., Joukhadar, S., 1983. Unmyelinated nociceptive units in two skin areas of the rat. Brain Res. 267, 81^92. Garell, P.C., McGillis, S.L.B., Greenspan, J.D., 1996. Mechanical response properties of nociceptors innervating feline hairy skin. J. Neurophysiol. 75, 1177^1189. Gentle, M.J., 1989. Cutaneous sensory a¡erents recorded from the nervus intramandibularis of Gallus gallus var domesticus. J. Comp. Physiol. A 164, 763^774. Gentle, M.J., 1991. The acute e¡ects of amputation on peripheral trigeminal a¡erents in Gallus gallus var domesticus. Pain 46, 97^103. Gentle, M.J., 1992. Ankle joint (artc. intertarsalis) receptors in domestic fowl. Neuroscience 49, 991^1000. Gentle, M.J., 1997. Sodium urate arthritis : e¡ects on the sensory properties of articular a¡erents in the chicken. Pain 70, 245^251. Gentle, M.J., Corr, S.A., 1995. Endogenous analgesia in the chicken. Neurosci. Lett. 201, 211^214. Gentle, M.J., Hill, F.L., 1987. Oral lesions in the chicken: behavioural responses following nociceptive stimulation. Physiol. Behav. 40, 781^783. Gentle, M.J., Hunter, L.N., 1990. Physiological and behavioural responses associated with feather removal in Gallus gallus var domesticus. Res. Vet. Sci. 50, 95^101. Gentle, M.J., Thorp, B.H., 1994. Sensory properties of ankle joint capsule mechanoreceptors in acute monoarthritic chickens. Pain 57, 61^374. Gentle, M.J., Tilston, V.L., 1999. Reduction in peripheral in£ammation by changes in attention. Physiol. Behav. 66, 289^292.
NSC 5159 26-9-01
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M. J. Gentle et al.
Gentle, M.J., Hunter, L.N., Corr, S.A., 1997. E¡ects of caudolateral neostriatal ablations on pain-related behaviour in the chicken. Physiol. Behav. 61, 493^498. Gentle, M.J., Hunter, L.N., Waddington, D., 1991. The onset of pain related behaviours following partial beak amputation in the chicken. Neurosci. Lett. 128, 113^116. Georgopoulos, A.P., 1976. Functional properties of primary a¡erent units probably related to pain mechanisms in primate glabrous skin. J. Neurophysiol. 39, 71^83. Georgopoulos, A.P., 1977. Stimulus-response relations in high-threshold mechanothermal ¢bers innervating primate glabrous skin. Brain Res. 128, 547^552. Gottschaldt, K.M., 1985. Structure and function of avian somatosensory receptors. In: King, A.S., McLelland, J. (Eds.), Form and Function in Birds, Vol. 3. Academic Press, London, pp. 375^461. Handwerker, H.O., Anton, F., Reeh, P.W., 1987. Discharge patterns of a¡erent cutaneous nerve ¢bers from the rat's tail during prolonged noxious mechanical stimulation. Exp. Brain Res. I 65, 493^504. Kenton, B., Kruger, L., Woo, M., 1971. Two classes of slowly adapting mechanoreceptor ¢bres in reptile cutaneous nerve. J. Physiol. 212, 21^44. Koltzenburg, M., Lewin, G.R., 1997. Receptive properties of embryonic chick sensory neurons innervating skin. J. Neurophysiol. 78, 2560^2568. Kumazawa, T., Perl, E.R., 1977. Primate cutaneous sensory units with unmyelinated (C) ¢bers. J. Neurophysiol. 40, 1325^1338. LaMotte, R.H., Thalhammer, J.G., Robinson, C.J., 1983. Peripheral neural correlates of magnitude of cutaneous pain and hyperalgesia: a comparison of neural events in monkey with sensory judgements in humans. J. Neurophysiol. 50, 1^26. LaMotte, R.H., Thalhammer, J.G., Torebjo«rk, H.E., Robinson, C.J., 1982. Peripheral neural mechanisms of cutaneous hyperalgesia following mild injury by heat. J. Neurosci. 2, 765^781. Leonard, R.B., 1985. Primary a¡erent receptive ¢eld properties and neurotransmitter candidates in a vertebrate lacking unmyelinated ¢bres. Prog. Clin. Biol. Res. 176, 135^145. Lewis, T., Pochin, E.E., 1937. The double pain response of the human skin to a single stimulus. Clin. Sci. 3, 67^76. Lucas, A.M., Stettenheim, P.R., 1972. Avian Anatomy. Integument Part II. United States Department of Agriculture, Washington. Lynn, B., 1979. The heat sensitization of polymodal nociceptors in the rabbit and its independence of local blood supply. J. Physiol. 287, 493^507. Lynn, B., 1994. The ¢bre composition of cutaneous nerves and the classi¢cation and response properties of cutaneous a¡erents, with particular reference to nociception. Pain Rev. 1, 172^183. Lynn, B., Carpenter, S.E., 1982. Primary a¡erent units from the hairy skin of the rat hind limb. Brain Res. 238, 29^43. Marin-Burgin, A., Reppenhagen, S., Klusch, A., Wendland, J.R., Petersen, M., 2000. Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitive. Eur. J. Neurosci. 12, 3560^3566. Martin, A.R., Wickelgren, W.O., 1971. Sensory cells in the spinal cord of the sea lamprey. J. Physiol. 212, 65^83. Matthews, G., Wickelgren, W.O., 1978. Trigeminal sensory neurons of sea lamprey. J. Comp. Physiol. 123, 329^333. Meyer, R.A., Campbell, J.N., 1981. Myelinated nociceptive a¡erents account for the hyperalgesia that follows a burn to the hand. Science 213, 1527^1529. Meyer, R.A., Campbell, J.N., Raja, S.N., 1985. Peripheral neural mechanisms of cutaneous hyperalgesia. In: Fields, H.L., Dubner, R., Cervero, F. (Eds.), Advances in Pain Research and Therapy. Raven Press, New York, pp. 53^71. Meyer, R.A., Davis, K.D., Cohen, R.H., Treede, R.D., Campbell, J.N., 1991. Mechanically insensitive a¡erents (MIAs) in cutaneous nerves of monkey. Brain Res. 561, 252^261. Nagy, I., Rang, H., 1999. Noxious heat activates all capsaicin-sensitive and also a sub-population of capsaicin-insensitive dorsal root ganglion neurons. Neuroscience 88, 995^997. Nagy, I., Rang, H., 2000. Comparison of currents activated by noxious heat in rat and chicken primary sensory neurons. Reg. Peptides 96, 3^6. Necker, R., Reiner, B., 1980. Temperature-sensitive mechanoreceptors, thermoreceptors and heat nociceptors in the feathered skin of pigeons. J. Comp. Physiol. A 135, 201^207. Proske, U., 1969. Vibration-sensitive mechanoreceptors in snake skin. Exp. Neurol. 23, 187^194. Roumy, M., Leitner, L.M., 1973. Activites a¡erentes provenant de bec superieur de la poule domestique. C.R. Acad. Sci. Paris 277, 1791^1794. Rundall, K.M., 1947. X-ray studies of the distribution of protein chain types in the vertebrate epidermis. Biochim. Biophys. Acta 1, 549^563. Snow, P.J., Plenderleith, M.B., Wright, L.L., 1993. Quantitative study of primary sensory neurone populations of three species of elasmobranch ¢sh. J. Comp. Neurol. 334, 97^103. Spray, D.C., 1976. Pain and temperature receptors of anurans. In: Llinas, R., Precht, W. (Eds.), Frog Neurobiology. Springer Verlag, Berlin, pp. 607^628. Spearman, R.I.C., 1971. Integumentary system. In: Bell, D.J., Freeman, B.M. (Eds.), Physiology and Biochemistry of the Domestic Fowl. Academic Press, London, pp. 603^620. Stevens, C.W., 1992. Alternatives to the use of mammals for pain research. Life Sci. 50, 901^912. Simino¡, R., 1968. Quantitative properties of slowly adapting mechanoreceptors in alligator skin. Exp. Neurol. 21, 290^306. Simino¡, R., Kruger, L., 1968. Properties of reptilian cutaneous mechanoreceptors. Exp. Neurol. 20, 403^414. Torebjo«rk, H.E., Schmelz, M., Handwerker, H.O., 1996. Functional properties of human cutaneous nociceptors and their role in pain and hyperalgesia. In: Belmonte, C., Cervero, F. (Eds.), Neurobiology of Nociceptors. Oxford University Press, Oxford, pp. 349^369. Treede, R.D., Meyer, R.A., Campbell, J.N., 1998. Myelinated mechanically insensitive a¡erents from monkey hairy skin: heat-response properties. J. Neurophysiol. 80, 1082^1093. Treede, R.D., Meyer, R.A., Raja, S.N., Campbell, J.N., 1995. Evidence for two di¡erent heat transduction mechanisms in nociceptive primary a¡erents innervating monkey skin. J. Physiol. 483, 747^758. White, D.M., Levine, J.D., 1991. Di¡erent mechanical transduction mechanisms for the immediate and delayed responses of rat C-¢ber nociceptors. J. Neurophysiol. 66, 363^368. (Accepted 21 July 2001)
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