Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. 2003 Jan 1; 546(Pt 1): 251–265.
Published online 2002 Nov 1. doi:  10.1113/jphysiol.2002.025023
PMCID: PMC2342464

Organisation of sensitisation of hind limb withdrawal reflexes from acute noxious stimuli in the rabbit


Spatial aspects of central sensitisation were investigated by studying the effects on three hind limb withdrawal reflexes of an acute noxious stimulus (20 % mustard oil) applied to a number of locations around the body in decerebrate and in anaesthetised rabbits. Reflex responses to electrical stimulation of the toes were recorded from the ankle flexor tibialis anterior (TA) and the knee flexor semitendinosus (ST), whereas responses to stimulation of the heel were recorded from the ankle extensor medial gastrocnemius (MG). In non-spinalised, decerebrated, pentobarbitone-sedated preparations, flexor reflexes were facilitated significantly from sites on the plantar surface of the ipsilateral foot but were either inhibited or unaffected by stimulation of sites away from this location. The heel–MG reflex was facilitated from the ipsilateral heel and was inhibited from a number of ipsilateral, contralateral and off-limb sites. In decerebrated, spinalised, pentobarbitone-sedated animals, mustard oil applied to any site on the ipsilateral hind limb enhanced both flexor reflexes, whereas the MG reflex was enhanced only after stimulation at the ipsilateral heel and was inhibited after stimulation of the toe tips or TA muscle. Mustard oil on the contralateral limb had no effect on any reflex. In rabbits anaesthetised with pentobarbitone and prepared with minimal surgical interference, the sensitisation fields for the heel–MG and toes–TA reflexes were very similar to those in non-spinal decerebrates whereas that for toes–ST was more like the pattern observed in spinalised animals. In no preparation was sensitisation or inhibition of reflexes related to the degree of motoneurone activity generated in direct response to the sensitising stimulus. This study provides for the first time a complete description of the sensitisation fields for reflexes to individual muscles. Descending controls had a marked effect on the area from which sensitisation of flexor reflexes could be obtained, as the sensitisation fields for the flexor reflexes evoked from the toes were larger in spinalised compared to decerebrated, non-spinalised animals. The intermediate sizes of sensitisation fields in anaesthetised animals suggests that the area of these fields can be dynamically controlled from the brain. On the other hand, the sensitisation field for the heel–MG reflex varied little between preparations and appears to be a function of spinal neurones.

Noxious stimuli induce limb withdrawal reflexes that have evolved to minimise tissue damage (Sherrington, 1910). Until recently it was considered that limb withdrawal was based on a monolithic response in which all flexor muscles ipsilateral to the stimulus were excited and all extensors inhibited. Building on the early work of Hagbarth (Hagbarth, 1952), Schouenborg and colleagues showed that the recruitment of muscles into hind limb withdrawal reflexes in the rat is in fact organised such that noxious stimuli activate those muscles that are best situated to move the limb directly away from the stimulus (Schouenborg & Kalliomaki, 1990; Schouenborg et al. 1994) and inhibit those muscles that would produce a movement towards the stimulus (Weng & Schouenborg, 1996). This type of organisation has also been seen in rabbit (Clarke et al. 1989), man (Andersen et al. 1999) and cat (Levinsson et al. 1999a).

Withdrawal reflexes show strong central sensitisation after mild noxious stimulation of the foot (Woolf, 1983; Woolf & Wall, 1986; Clarke et al. 1992b). The present study has been designed to investigate whether the sensitisation of reflexes shows the same sort of spatial organisation as the reflexes themselves. That this may be so was initially suggested by studies from this laboratory showing that in decerebrated, spinalised rabbits, reflexes evoked in the ankle extensor medial gastrocnemius (MG) by activation of afferents from the heel were facilitated after noxious stimulation at that site but inhibited by noxious stimulation at the toes (Taylor et al. 1990; Clarke et al. 1992b). As MG is an ankle extensor that acts to shift weight from the heel to the toes, these results are consistent with the modular organisation of reflexes described above. However, experiments on reflexes evoked by stimulation at the toes in the knee flexor semitendinosus (ST), which acts to remove the entire foot from contact with the ground, and the ankle flexor tibialis anterior (TA), which shifts weight from the toes to the heel, showed no differences between the effects of noxious stimulation at the heel and the toes (Clarke & Harris, 2001). A single small-scale study in non-spinalised animals showed that the heel–MG reflex showed the same pattern of excitation and inhibition as seen in spinal preparations, but that the toes–TA reflex was not potentiated from the heel (Clarke et al. 2001). The collected data from these experiments, based on mustard oil stimulation of just two sites, indicate that there is some spatial organisation of central sensitisation of reflexes to individual muscles but do not allow us to define the principles upon which the organisation is based. Furthermore, all of these studies were performed in preparations with extensive surgical exposure of the sciatic nerve, and it is perfectly possible that the massive nociceptive bombardment arising from the wound could alter the reflexes and the way they respond to sensitising stimuli.

The present study addresses the question of spatial organisation of central sensitisation in a number of ways. A more comprehensive view of the relationship between the location of a sensitising stimulus and the reflex adaptations to it has been obtained by investigating the effects of a wide range of stimulation sites on the ipsilateral hind limb and other parts of the body, on three withdrawal reflexes recorded at the same time, to whit heel–MG, toes–ST and toes–TA. Knowledge of the movements generated by the effector muscles informed our choice of the range of locations to which the sensitising stimuli were applied in the ipsilateral hind limb (see Methods). Sites away from the ipsilateral hind limb were also studied in the expectation that they would give rise predominantly to inhibition (Taylor et al. 1991). The influence of descending systems on the sensitisation per se (Urban & Gebhart, 1999) and upon its organisation (Schouenborg et al. 1992; Clarke et al. 2001) has been determined by performing the primary part of the study in non-spinalised decerebrated animals and comparing the results of these experiments with a complementary investigation in spinalised decerebrates. The possible confounding effects of surgery have been controlled for by recording reflexes as EMG signals from percutaneous electrodes (i.e. no dissection of the limbs), and by performing a final set of experiments in anaesthetised animals prepared with minimal surgical interference (Ogilvie et al. 1999). In experiments on spinalised and anaesthetised animals, sensitisation sites were selected to provide optimum comparison with the data obtained in the primary study.

The process of central sensitisation probably underlies the development of chronic pain states (Coderre & Katz, 1997; Clarke, 2000), yet the relationship between the location of an injury and the spread of the central changes that follow from it has never been investigated. The present study will begin to address this issue. Some of the data described below have been reported previously in abstracts (Harris et al. 2001; Harris & Clarke, 2002a,b).


Studies were performed under the UK Animals (Scientific Procedures) Act of 1986. Experiments were performed on 93 rabbits of mixed strains and either sex, weighing between 1.6 and 3.6 kg. All animals were sedated with ketamine sulphate (Fort Dodge Animal Health, UK, 50 mg i.m.) prior to anaesthesia.

Decerebrated preparations

Animals that were to be decerebrated (n = 76) were anaesthetised with i.v. methohexitone sodium (Brietal, Eli Lilly, average dose 14 mg kg−1, n = 66) or propofol (Diprivan, ICI, average dose 15 mg kg−1, n = 10) given over 15 min. The trachea was cannulated and anaesthesia continued using halothane (0.5-3.5 %) delivered in N2O:O2 (70:30). The left carotid artery and jugular vein were cannulated to allow measurement of arterial blood pressure and administration of drugs, respectively. At this point, 28 animals were spinalised at the thoracolumbar junction. The L1 vertebra was removed and the cord anaesthetised with ca 100 μl 2 % lignocaine solution (C-Vet). A 3-4 mm section of cord was then aspirated and the space filled with cotton wool moistened with lignocaine solution. All animals were then decerebrated by suction to the precollicular level to render the preparations insensate and anaesthesia discontinued.

All decerebrated animals were given a sub-anaesthetic infusion of pentobarbitone sodium (Sagatal, Rhone Merieux), 1 mg ml−1 in 100 mm d-glucose-100 mm NaHCO3 solution, infused i.v. at a mean rate of 2.6 mg kg−1 h−1 (i.e. a total fluid infusion of ca 5 ml h−1).

Preparations were allowed to respire spontaneously. End tidal CO2 was measured at intervals but deviations from normal could not be remedied. It was not unusual for these preparations to hyperventilate and the range of end tidal CO2 recorded was between 2.0 and 5.0 %. Mean (± s.e.m.) end tidal CO2 was 3.3 ± 0.1 and 3.1 ± 0.2 % in non-spinal and spinal animals, respectively (not significantly different, Student's t test, P > 0.3). ECG was recorded from needle electrodes placed either side of the chest and the signal used to trigger a Neurolog NL 253 rate meter to give a record of heart rate. Core temperature was maintained at 37-38 °C by the action of a thermostatically controlled heating blanket. Experiments were terminated by i.v. injection of 2 ml saturated KCl solution.

Anaesthetised preparations

Seventeen rabbits were anaesthetised with pentobarbitone sodium (diluted to 30 mg ml−1 in Ringer solution, mean initial dose 32 mg kg−1i.v.). All invasive surgery was carried out after infiltration of the relevant tissues with 2 % lignocaine solution. The trachea, left carotid artery and jugular vein were cannulated as described above. Anaesthesia was then maintained by a continuous i.v. infusion of pentobarbitone (6 mg ml−1 diluted in 100 mm d-glucose, 100 mm NaHCO3 solution) at a mean rate of 13 mg kg−1 h−1 (range 5-19 mg kg−1 h−1) during the recording period. No other surgery was performed. These preparations were artificially ventilated on room air supplemented with O2. Blood pressure, heart rate, core temperature and end tidal CO2 were all monitored as described above. In these preparations ventilation was controlled and CO2 was held between 3.5 and 4.5 % (mean 3.8 ± 0.1 %). Experiments were terminated by giving an overdose of anaesthetic followed by 2 ml saturated KCl solution.

Stimulation and recording

Reflexes were evoked by electrical stimulation of the plantar skin of the foot at the heel (the small bald patch at the calcaneus) and at the metatarsophalangeal joints of the middle two toes using paired, stainless steel 23 gauge needle electrodes separated by 4 mm. The electrodes were sometimes positioned slightly central to these locations to allow the skin to be stimulated with mustard oil at the relevant points. Electrical stimuli were constant current pulses of 1 ms duration delivered from AMPI Isoflex stimulators. The stimulus was set to a multiple (between 1.5 and 4 times) of the threshold value for evoking reflexes. The need to record a measurable reflex response with some room to increase in size was the determinant of the multiple used in each experiment. In decerebrated, non-spinalised rabbits it was often not possible to evoke reflexes in the flexor muscles with single shocks up to 10 mA. Therefore in the majority of these preparations, the toes were stimulated with triple pulses delivered at 250 Hz. To allow for comparisons with experiments in which single shocks were given, the threshold for evoking reflexes in animals requiring triple shocks was recorded as 10 mA (i.e. the highest possible single stimulus). Stimuli were delivered in blocks of eight at 1 Hz, applied alternately to the heel and the toes at 2 min intervals.

Reflex responses were recorded as compound EMG signals from the medial gastrocnemius, tibialis anterior and semitendinosus muscles ipsilateral to the stimulating electrodes, using paired, percutaneous, varnish-insulated copper wire electrodes. On the basis of previous studies (Clarke et al. 1989, 1992a), only MG reflexes were recorded in response to heel stimulation and only TA and ST reflexes were recorded after stimulation at the toes. The signals were amplified 1000-5000 times, filtered between 1 and 6 kHz, and fed to a CED 1401 interface where the responses to each 8-stimulus block could be averaged and integrated using SIGAVG v. 6.3 (CED) running on a personal computer. Amplified signals were also put through a second set of filters (0.5-5 kHz) and fed to a spike discriminator that was set to capture all clearly visible action potentials, using audio output to assist in determining the correct level at which to set the discriminator. This record provided an indication of the level of ongoing activity within each muscle overall: no attempt was made to discern activity of single motor units. The output from these devices was fed to a CED micro1401 interface connected to a second computer running Spike2 for Windows v. 3 (CED). This program was used to create records of mean frequency of activity in each of the three muscles, in addition to providing continuous recording of arterial blood pressure and heart rate.

Conditioning stimuli

The noxious conditioning stimulus used was 100 μl allyl-isothiocyanate (mustard oil, Aldrich) diluted to 20 % in liquid paraffin. For superficial sites this was applied directly to the shaved skin from a de-pointed 19 gauge needle. For deep sites, the oil was injected into the relevant tissue through a 25 gauge needle. No attempt was made to remove the oil once it had been applied. Up to eight conditioning stimuli were applied in any one experiment, separated by intervals of at least 63 min. No site was stimulated more than once in an experiment, and the order in which locations were stimulated was randomised as far as possible. Injection of mustard oil into the MG muscle or the ankle joint occasionally stopped all reflexes working, an effect that seemed to arise from conduction block in nerves passing close to the stimulated site. For that reason, these stimuli were usually given at the end of an experiment.

Mustard oil was applied to 20 different locations in decerebrated, non-spinalised preparations (Table 1). In spinalised animals no stimuli were applied above the level of spinal section and only 11 sites were stimulated altogether (Table 1). In anaesthetised animals, stimuli were applied to nine different locations (Table 1). The sites for the spinalised and anaesthetised preparations were chosen to allow an optimum level of comparison with decerebrated, non-spinalised animals while avoiding an excessive number of experiments.

Table 1
Summary of sites stimulated in each preparation, and the number of times each site received a sensitising stimulus, with notes on how the mustard oil was applied in each case

Statistical analysis

The effects of mustard oil on reflexes were assessed by expressing reflexes as the percentage of the mean of the three responses recorded immediately prior to the stimulus. Statistical analysis of changes in the voltage-time integral (area) of reflex responses was carried out using Friedman's ANOVA on ranks. The time to recovery after a conditioning stimulus was also measured. This was taken as the time when reflexes had recovered to within 2 standard deviations of the mean pre-stimulus control level for two successive readings, with cut-off values of 61 min for the heel–MG reflex and 63 min for the flexor reflexes. Changes in background activity of muscles were assessed using similar non-parametric analyses, based on firing rates averaged over 1 min time bins. Blood pressure data were analysed using standard parametric tests, again based on averaging values over 1 min intervals. Comparisons between stimulus sites and preparations were made using Mann-Whitney U tests, Wilcoxon's test or Kruskal-Wallis ANOVA. Data were analysed using GraphPad InStat v.3.


Comparisons between preparations

Considering the decerebrated, sedated preparations, spinalised animals showed significantly lower arterial blood pressure, higher levels of ongoing activity, lower thresholds for evoking reflexes and larger absolute reflex responses (despite the use of lower stimulus intensities) than their non-spinalised counterparts (Table 2). Anaesthetised animals showed characteristics that were an amalgam of those seen in the other two preparations: blood pressure and thresholds for evoking reflexes from the toes were ‘spinal’ (i.e. not significantly different from values found in spinalised animals but significantly lower than those observed in non-spinalised preparations), whereas ongoing activity, thresholds for evoking reflexes from the heel and absolute reflexes were ‘non-spinal’ (Table 2).

Table 2
Baseline (i.e. before any mustard oil was applied) data for (top) arterial blood pressure and ongoing activity, and (bottom) thresholds for evoking reflexes and absolute sizes of evoked reflexes

Ongoing activity was generally low and not significantly different between muscle nerves in any preparation (Kruskal-Wallis ANOVA, P > 0.2). Thresholds for evoking reflexes from the heel were significantly lower than those for the toes in non-spinalised animals (Wilcoxon's test, P < 0.0001), and higher in spinalised and anaesthetised animals (Wilcoxon, P < 0.04, Table 2). Owing to the way stimulus intensities were set, the only meaningful comparisons between the absolute sizes of reflexes are between the responses of the two flexor muscles. TA reflexes to toe stimulation were significantly larger than those of ST in the non-spinalised and anaesthetised preparations (P < 0.001, Wilcoxon's test), but the difference between the two in spinalised preparations did not reach significance (Table 2, Wilcoxon, P > 0.05).

Responses to mustard oil

In the sedated, decerebrated, non-spinalised preparations, application of mustard oil to deep and mid-line structures often gave rise to vigorous movements in all four limbs and was usually followed by increases in blood pressure, augmented background firing of all three muscle nerves and a range of changes in evoked reflex responses as detailed below. Spontaneous movements of the same type, which were not infrequent, were associated with similar changes in blood pressure and background firing, but were followed by inhibition, for between 1 and 11 min, of all three reflexes. Thus it can be deduced that the changes in reflexes described below were not secondary to the movements generated.

Changes in ongoing activity

The MG muscle showed significantly enhanced activity after application of mustard oil to the ipsilateral heel in all three preparations (Friedman's ANOVA, P < 0.04, Figs 1--4),4), with peak median increases over pre-mustard values of 19 (IQR 1-30), 9 (IQR 4-16) and 1 (IQR 0-50) spikes s−1 in non-spinal, spinal and anaesthetised animals, respectively. In sedated decerebrates the increased activity persisted for 4-5 min, but in anaesthetised preparations it lasted for just 1 min. In non-spinal decerebrates and anaesthetised animals, mustard oil evoked responses of similar magnitude and duration to those obtained from the heel when it was applied to the mid-sole position. In non-spinalised decerebrates, MG also showed transient (1 min) responses after application of mustard oil to a wide range of other sites from all around the body (Fig. 4). These sites were predominantly those from which strong movements were evoked (i.e. deep and mid-line structures, see above).

Figure 1
Upper panel, reflexes evoked in medial gastrocnemius (MG) from stimulation at the heel, and semitendinosus (ST) and tibialis anterior (TA) from stimulation at the toes before and after application of mustard oil to the heel in a decerebrated, sedated ...
Figure 4
Effects of mustard oil on background activity in MG (top row), ST (middle row) and TA (bottom row) muscles in decerebrated, sedated, non-spinalised rabbits (left column), decerebrated, sedated, spinalised rabbits (middle column) and in anaesthetised rabbits ...

Ongoing activity in ST increased after mustard oil stimulation of all ipsilateral plantar sites other than the toe tips in all preparations (Friedman's ANOVA, P < 0.03, Figs 1--4).4). From these locations the largest effects were always obtained from the mid-sole position, which gave median increases of 29 (IQR 5-69), 58 (IQR 22-140) and 9 (IQR 0-16) spikes s−1 over pre-mustard levels in non-spinal, spinal and anaesthetised preparations respectively. Responses persisted for no more than 1 min in non-spinalised and anaesthetised animals (e.g. Fig. 1 and Fig. 3), but lasted for up to 10 min in spinalised preparations (e.g. Fig. 2). In non-spinalised preparations ST ongoing activity increased transiently after application of mustard oil to some deep tissues on the ipsilateral limb and to all of the mid-line structures, i.e. those tissues from which whole body movements were activated. In spinalised preparations ST responded vigorously to mustard oil applied to almost every part of the ipsilateral hind limb.

Figure 2
As for Fig. 1, from a spinalised, decerebrated, sedated rabbit. The voltage scale is 200 μV for heel–MG and toes–TA reflexes and 100 μV for toes–ST.
Figure 3
As for Fig. 1 from a pentobarbitone-anaesthetised rabbit with minimal surgical interference.

TA showed broadly similar patterns of responses to mustard oil stimuli to those seen in ST (Fig. 4). The main differences were that responses to stimulation at the heel were absent in non-spinal and anaesthetised preparations (Fig. 1 and Fig. 3) and weak in spinalised animals (Fig. 2). The duration of increased activity in spinalised animals was rather less than for ST at around 4 min, and this muscle showed no significant increases in background activity to any stimulus in the anaesthetised animals (Fig. 4).

Arterial blood pressure

Mustard oil induced significant increases in blood pressure from almost all stimulated sites in decerebrated, non-spinalised and anaesthetised animals (repeated measures ANOVA, P < 0.05, Fig. 5). These increases were of the order of 4-7 mmHg over control in non-spinal decerebrates and 8-10 mmHg in anaesthetised preparations, with a duration of approximately 10-20 min (Fig. 1 and Fig. 3). Application of the sensitising stimulus to the snout induced larger increases in blood pressure than other sites (mean change 16 ± 3 and 25 ± 3 mmHg in non-spinal and anaesthetised animals, respectively), but the difference was significant only in non-spinal animals (ANOVA followed by Tukey-Kramer test, P < 0.05). Mustard oil caused no significant changes in blood pressure in spinalised preparations (repeated measures ANOVA, P > 0.05).

Figure 5
Changes in blood pressure induced by application of mustard oil in decerebrated, sedated, non-spinalised rabbits (left), decerebrated, sedated, spinalised rabbits (middle) and in anaesthetised rabbits (right)

Adaptive changes in reflexes after mustard oil

The data reported below show consensual results from 6-12 experiments for each mustard oil treatment. A significant change in a reflex indicates the response that was most likely to follow a particular stimulus, but it may not have occurred in all animals. The figures quoted for peak changes in reflexes include data from all experiments and were taken 3 min after mustard oil treatment for flexor reflexes and 5 min after for the MG reflex. Figures for duration of mustard oil-induced effects include only those experiments where the predominant change in reflex response occurred. Some sites from which no overall effect was obtained, such as the ipsilateral ankle joint in decerebrated non-spinalised animals (see Fig. 7), represent a balance between several animals showing inhibition and others showing potentiation. Stimulation of forelimb sites failed to generate any significant effects in non-spinal decerebrates (Fig. 6) so they were not investigated further.

Figure 6
Changes in the heel–MG (top row), toes–ST (middle row) and toes–TA (bottom row) reflexes elicited by application of mustard oil to sedated, decerebrated, non-spinalised animals (left column), sedated, decerebrated, spinalised animals ...
Figure 7
Effects of mustard oil applied to the ipsilateral ankle joint on the heel–MG (top), toes–ST (middle) and toes–TA (bottom) reflexes in sedated, decerebrated, non-spinalised rabbits (open circles), sedated, decerebrated, spinalised ...

Heel–MG reflex

The heel–MG reflex was facilitated after application of mustard oil to the ipsilateral heel in all three preparations (Friedman's ANOVA, P < 0.05, Fig. 1--33 and Fig. 6). The peak increases were to medians of 250 % (IQR 133-524 %), 119 % (IQR 91-151 %) and 493 % (IQR 195-791 %) of pre-mustard values in non-spinal, spinal and anaesthetised preparations, respectively. The corresponding median durations of the enhancement were 41 (IQR 19-61), 61 (IQR 39-61) and 45 (IQR 39-61) min. Potentiation of the reflex after stimulation of the heel in spinalised animals was slow in onset and reached a significantly lower peak value than was seen in the anaesthetised preparations (Kruskal-Wallis ANOVA, P < 0.04, followed by Dunn's test, P < 0.05).

This reflex was the only one to show any inhibition after mustard oil in spinalised preparations, or inhibition to < 70 % control from sites on the ipsilateral hind limb (Fig. 6). In sedated, non-spinalised decerebrates the MG responses were suppressed after stimulation of almost all superficial ipsilateral hind limb, contralateral hind limb and mid-line sites. There were no significant differences between the maximum decreases evoked from individual sites (Kruskal-Wallis ANOVA, P > 0.8), but stimulation at the ipsilateral plantar metatarsophalangeal (MT) area and knee, the contralateral ankle joint and the snout reduced MG reflexes to < 50 % pre-mustard oil levels (Fig. 6). Median duration of inhibition was between 13 and 61 min. Stimulation of the dorsal side of the metatarsophalangeal area resulted in a biphasic change in MG responses, with an initial inhibition (to a median of 51 %, IQR 33-93 %) superseded, after intervals of between 9 and 41 min, by facilitation. Peak enhancement was to a median of 210 % (IQR 113-331 %) of pre-stimulus levels, 53 min after the stimulus.

Fewer sites evoked inhibition of heel–MG reflexes in spinal and anaesthetised preparations (Fig. 6). In spinal animals, the reflex was depressed only after mustard oil application to the distal parts of the ipsilateral hind paw, while in anaesthetised animals stimulation of deep tissues either side of the body, and the snout generated suppression of this response. There were few statistically significant differences between maximum changes evoked from individual sites compared between preparations: the responses to stimulation of the dorsum of the ankle and the contralateral heel were significantly different between non-spinal and the other two preparations (Kruskal-Wallis ANOVA, P < 0.04, followed by Dunn's test P < 0.05), and the effects of stimulation at the knee were significantly different between spinal and non-spinal decerebrates (Mann-Whitney, P < 0.02).

Toes–ST reflex

ST responses to stimulation at the toes were potentiated from all ipsilateral plantar sites tested in all three preparations (Friedman's ANOVA, P < 0.04, Fig. 1--33 and Fig. 6). The ranges of median increases at 3 min after application of mustard oil were 145-197, 182-203 and 264-587 % of pre-mustard oil levels in non-spinal decerebrates, spinal decerebrates and anaesthetised animals, respectively. Corresponding median durations of enhancement were 19-35, 21-59 and 25-51 min. Comparing effects evoked from individual sites, there were no significant differences between preparations (Kruskal-Wallis ANOVA, P > 0.1).

In non-spinal decerebrates, the ipsilateral MG muscle was the only other site from which a significant increase was generated in the toes–ST reflex (Fig. 6), and this effect was weak (median increase at 5 min 112 %, IQR 100-126 % of pre-mustard levels). On the other hand, in spinalised animals stimulation of all sites in the ipsilateral hind limb gave significant enhancement of this reflex (Friedman's ANOVA, P < 0.05, Figs 2, ,66 and and7).7). There were no significant differences between the effects evoked from individual sites (Kruskal-Wallis ANOVA, P > 0.4). The enhancements evoked from the dorsum of the ankle, ankle joint (Kruskal-Wallis ANOVA, P < 0.02, followed by Dunn's test, P < 0.05) and the knee (Mann-Whitney, P < 0.04) were significantly greater than the effects observed in non-spinal decerebrates. Anaesthetised preparations showed a very similar pattern of mustard oil-evoked changes in the toes–ST reflex from stimulation of ipsilateral sites to that observed in spinalised animals, with the exception that the MG muscle generated no significant effects (Fig. 6). Only the effect elicited by stimulation at the ankle joint was significantly different between the two preparations (Kruskal-Wallis ANOVA, P < 0.04 followed by Dunn's test, P < 0.05, Fig. 7).

The toes–ST reflex was inhibited only from stimulation of mid-line structures (the tail tip and the snout) in non-spinal decerebrates (Fig. 6). The effects were weak (median decreases at 3 min to 87 %, IQR 75-88 % and 79 %, IQR 65-94 %, respectively) and lasted for 13 and 25 min (median duration).

Toes–TA reflex

This reflex was significantly augmented after application of mustard oil to ipsilateral distal plantar sites and the mid-sole position in all three preparations (Friedman's ANOVA, P < 0.04, Fig. 6). The range of median increases were to 123-210, 214-457 and 216-343 % of pre-mustard oil levels in non-spinal decerebrates, spinal decerebrates and anaesthetised animals, respectively. Corresponding median durations of enhancement were 47-63, 17-63 and 41-63 min. Stimulation of the plantar MT area induced a significantly greater increase in TA reflexes in spinal compared to non-spinal animals (Mann-Whitney, P < 0.01), but there were no differences between preparations in respect of the other sites (Kruskal-Wallis ANOVA, P > 0.05). Unlike the other two reflexes, toes–TA was not facilitated (Friedman's ANOVA, P > 0.1) after mustard oil stimulation at the heel in either non-spinal decerebrates (median at 3 min 98 % of pre-mustard values, IQR 47-115 %, Fig. 1) or anaesthetised preparations (median at 3 min 117 % of pre-mustard values, IQR 89-153 %, Fig. 3).

The toes–TA reflex was, like toes–ST, significantly (Friedman's ANOVA, P < 0.03) enhanced when mustard oil was applied to any of the locations on the hind limb in spinalised preparations (Figs 2, ,66 and and7),7), including the heel. After stimulation at this site, TA responses were enhanced to a median of 244 % (IQR 129-274 %) of pre-mustard values, and the median duration of the effect was 63 (IQR 21-63) min. This effect was significantly different from that observed in non-spinalised animals (Kruskal-Wallis ANOVA, P < 0.01, followed by Dunn's test) but not quite significantly different from the changes in anaesthetised preparations. Other ipsilateral sites from which significantly (Kruskal-Wallis ANOVA or Mann-Whitney test, P < 0.01) greater effects were obtained in spinalised compared to non-spinalised rabbits were the dorsum of the ankle, the knee, the ankle joint and the MG muscle (Fig. 6 and Fig. 7). There were no significant differences in the levels of enhancement evoked by mustard oil from individual ipsilateral sites (Kruskal-Wallis ANOVA, P > 0.3). Anaesthetised animals generally followed the non-spinal decerebrated pattern of effects, except that stimulation at the ankle joint induced significant facilitation of TA reflexes (Friedman's ANOVA, P < 0.001). This change was unusually slow in developing: the peak increase in reflexes, to a median of 194 % of pre-mustard values (IQR 114- 277 %), was not reached until 39 min after mustard oil had been applied (Fig. 7).

The toes–TA reflex was inhibited (Friedman's ANOVA, P < 0.05) after stimulation of most contralateral hind limb and midline sites in the non-spinal decerebrates (Fig. 6). The reflex was reduced to < 70 % pre-mustard levels after stimulation at the contralateral ankle joint and MG muscles, and the snout (median decreases were to 62 % of pre-mustard values, IQR 29-97 %, 64 %, IQR 38-89 % and 38 %, IQR 18-63 %, respectively, Fig. 6). The corresponding median durations of these effects were 31, 63 and 47 min. A weak, short duration inhibition (median decrease to 78 % of pre-mustard values, IQR 64-100, duration 13 min) was also evoked from mustard oil stimulation of the ipsilateral ankle joint (Fig. 7). Inhibition of this reflex was also obtained from off-limb sites in anaesthetised animals, notably the contralateral MG muscle and the snout (Fig. 6).


Differences between preparations: baseline data

In decerebrated, spinalised, sedated animals, the arterial blood pressure and the thresholds for evoking reflexes were predictably lower than in their non-spinalised counterparts. The lower blood pressure results from the loss of excitatory drive from the rostral ventrolateral medulla to the sympathetic preganglionic neurones of the spinal cord, whereas the enhanced excitability of reflex pathways arises from the removal of tonic descending inhibition (Eccles & Lundberg, 1959; Engberg et al. 1968a; Hillman & Wall, 1969; Handwerker et al. 1975; Duggan & Morton, 1988). The effects of spinalisation on the flexor reflexes were greater than those observed for the heel–MG reflex. This finding suggests that there is some differentiation in the tonic inhibition directed at reflexes evoked in MG versus those in flexors, with the latter being both more effective and more susceptible to inhibition by pentobarbitone. The possibility of differential descending control of cutaneous reflexes to extensor versus flexor muscles was raised many years ago (Holmqvist & Lundberg, 1961; Engberg et al. 1968b), but has received relatively little attention.

Effects of mustard oil

Mustard oil was able to sensitise reflexes in all three preparations, but the areas from which sensitisation could be obtained varied with each reflex and preparation. The investigators’ personal experience is that application of 100 μl 20 % mustard oil to the skin gives a mild to moderate stinging sensation that lasts for 2-3 min. In rats, this stimulus has been shown to be a selective activator of C-fibres (Woolf & Wall, 1986) and to induce reflex activity in motoneurones lasting for 3-5 min (Cook et al. 1987), consistent with the present findings. The evidence is that the mustard oil stimulus results in a moderate nociceptive barrage with a duration of just a few minutes. In most cases in the present study, the duration of changes in reflex responses induced by mustard oil exceeded 20 min and were often much longer, indicating that the effects of the conditioning stimulus outlasted the initial nociceptive input by a considerable margin.

Spatial organisation of sensitisation

The sites from which each reflex was sensitised irrespective of preparation were located in parts of the paw that the activated muscles would act to withdraw from contact with the ground: i.e. the heel for MG, the anterior portion of the plantar surface in the case of TA, the entire plantar surface for ST (Sherrington, 1910; Hagbarth, 1952; Schouenborg & Kalliomaki, 1990; Schouenborg & Weng, 1994; Andersen et al. 1999). In the rat the cutaneous excitatory receptive fields for limb muscles usually correspond to the area of skin withdrawn from contact with the ground when that muscle is contracted (Schouenborg & Weng, 1994), a level of organisation that is maintained by, but not entirely dependent upon, activity in descending pathways (Schouenborg et al. 1992). On the basis of the small sample of reflexes investigated in the present study, we would propose that the area withdrawn by a muscle represents a core from which withdrawal reflexes to that muscle can be sensitised irrespective of the state of activity in descending systems. As ST should act to withdraw dorsal as well as plantar parts of the hind paw, and yet was not facilitated from such sites in non-spinal decerebrates, we believe that the load-bearing parts of the paw have a special significance in terms of generating sensitisation.

Descending controls had a marked effect on the area from which sensitisation of flexor reflexes could be obtained, as both ST and TA reflexes evoked from the toes were potentiated after mustard oil stimulation of all tested sites on the ipsilateral hind limb in spinal preparations. This represented a large expansion of the ‘sensitisation’ fields for these reflexes relative to the decerebrated, non-spinal state. This is consistent with the alterations in receptive fields for the individual muscles reported in the rat (Schouenborg et al. 1992), albeit that the increase seen for ankle flexors is larger in the present study, and is reminiscent of the expansion of receptive fields of dorsal horn neurones that occurs when descending inhibition is blocked (Laird & Cervero, 1990). Anaesthetised animals showed patterns of sensitisation intermediate between the spinal and non-spinal decerebrates. The range of sensitising sites for toes–TA was close to that observed in non-spinal preparations, whereas the area from which toes–ST could be augmented was more like that found in spinalised animals.

Evidently descending pathways confer spatial organisation on the development of central sensitisation of flexor withdrawal reflexes to individual muscles. In rat the orderly development of receptive fields for individual muscles is learnt within a few days of birth and is dependent on the integrity of descending pathways (Holmberg & Schouenborg, 1996; Levinsson et al. 1999b). It has been reported that spinal long-term potentiation (a condition thought to be analogous to central sensitisation) is more difficult to evoke in preparations in which descending controls are intact (Sandkuhler & Liu, 1998; Gjerstad et al. 2001). The present data show that descending control of sensitisation is in fact permissive, in that where sensitisation is enabled, it is expressed to the same level as in spinal animals. The results obtained in anaesthetised animals, in which descending inhibition appears to be reduced relative to the decerebrates (see below), show that the area from which sensitisation is obtained can be dynamically controlled from the brain. It is possible that the strength or duration of the sensitising stimulus is also a key variable. The mustard oil stimulus is not particularly painful or persistent and it is conceivable that a more intense input, such as that provided by chronic inflammation or nerve damage, would break through the descending control to generate more global and/or chronic central changes than those observed in the present study.

An important question arises as to which of the three preparations used in the present study most closely approximates to that found in conscious preparations. Recordings from dorsal horn neurones in awake animals indicate that tonic descending control is strong and that it is reduced by anaesthesia (Collins, 1987; Collins et al. 1990; Herrero & Headley, 1995). From this viewpoint, the sedated, decerebrate non-spinal preparation appears to have most in common with the conscious state.

In contrast to the flexor reflexes, the sensitisation field of the heel–MG reflex hardly altered between preparations. With the interesting exception of the late response to stimulation of the dorsal surface of the toes, MG reflexes were enhanced only from the heel or nearby structures. Similar effects can be obtained after electrical stimulation of sural nerve afferents (Clarke et al. 1992b; Houghton et al. 2000), suggesting that the changes induced by mustard oil were primarily central in origin. There was no expansion of the sensitisation field for this reflex in spinal animals, indicating that the control of sites from which the heel–MG pathway can be sensitised is a function of the spinal cord rather than descending systems. Additionally, this reflex was the only one to be inhibited substantially from sites on the ipsilateral hind paw, particularly from the area around the toes as we have shown previously (Catley et al. 1984; Taylor et al. 1990; Bhandari et al. 1999). The likely reason for this is that contraction of MG shifts weight from the heel to the toes and would thus aggravate any injury at the latter site. The biphasic effect of stimulation of the dorsal surface of the toes on MG deserves further investigation. An argument can be made that MG could contribute to withdrawing this site (Sonnenborg et al. 2001), which may go some way to explaining this result. It would be interesting to see if this complex response is obtained also in spinal and anaesthetised preparations.

There are two particularly interesting features of sensitisation of the heel–MG reflex in spinal rabbits. Compared to the effect seen in non-spinal preparations, it was slow in onset, rather low in amplitude, and failed to occur at all in several animals. This is in marked contrast to the effects reported from spinalised preparations with extensive surgery on the hind limb, in which mustard oil applied to the heel augmented MG reflexes in a manner very similar to that seen in non-spinal animals in the present study (Clarke et al. 1992b; Houghton & Clarke, 1995). This is the only example we have found of a clear difference in effects based on the amount of surgical preparation, and also provides the only suggestion from the present study that supraspinal systems might contribute in a positive way to sensitisation, as has been suggested by others (Mansikka & Pertovaara, 1997; Coutinho et al. 1998; Urban et al. 1999; Urban & Gebhart, 1999). It would appear that surgical preparation of the limb, which we have shown to be the cause of tachykininergic tone in spinalised rabbits (Houghton et al. 1995), restores to the heel–MG pathway some factor that would otherwise be supplied by descending systems. This observation aside, where they can be directly compared the effects of mustard oil stimulation were entirely consistent with those obtained in animals in which the popliteal fossa was surgically exposed (Clarke et al. 1992b, 2001; Clarke & Harris, 2001), showing that the patterns of reflex adaptation observed were not unduly influenced by previous surgical history, or by decerebration per se.

Changes in ongoing activity

There was not always an obvious relationship between the ‘direct’ reflex actions of the mustard oil stimulus, as shown by changes in ongoing activity in the muscles, and sensitisation responses in reflexes. Mustard oil evoked large changes in ongoing activity in spinal animals and almost none in anaesthetised preparations yet, where it occurred, the degree of sensitisation of electrically evoked reflexes was much the same in the two preparations. In spinalised animals, TA motoneurones showed only a very small increase in activity after application of mustard oil to the heel, but a large response to stimulation of the toes. Nonetheless, sensitisation of electrically evoked reflexes from the two sites was indistinguishable. In non-spinal decerebrates, the MG muscle showed transient activation from a surprisingly wide range of sites scattered around the body, and yet sensitisation of the heel–MG reflex followed only from stimulation at the heel. Evidently activation of motoneurones per se is not a prerequisite for the development of sensitisation.

Inhibition from mustard oil stimulation

In the present study, sensitisation of reflexes was only ever obtained from structures in the ipsilateral hind limb, irrespective of the reflex studied or preparation used, and no effects were observed from off-limb sites in spinalised animals. The only significant effects observed from stimulation of structures away from the ipsilateral hind limb was inhibition. This is consistent with our earlier studies in which intense electrical conditioning stimuli applied to nerves in all four limbs of non-spinalised decerebrated rabbits resulted in inhibition of MG reflex responses (Taylor et al. 1991). Recent studies on rat dorsal horn neurones have reported similar phenomena (Gjerstad et al. 1999, 2000). Although other workers have reported enhancement of contralateral reflexes after acute noxious stimuli (Chen et al. 2000), we believe such changes are likely to be maladaptive. If injury to a limb requires adaptive enhancement of protective reflexes in the affected limb, possibly leading to a flexed posture, the other limbs must provide a stable platform for the animal and a generalised increase in threshold for other withdrawal reflexes would be one way of achieving this (Duggan & Morton, 1988).

It was difficult to inhibit the toes–ST reflex from any site. However, we believe that this was in part due to the small size of the reflex signal, which rendered decreases difficult to detect. The pattern of inhibition of the heel–MG and toes–TA reflexes from off-limb structures was fairly consistent, with stimulation of mid-line and contralateral hind limb sites most likely to result in suppression of reflexes. However, inhibition was less robust than facilitation and there was a lower chance of observing inhibitory effects in anaesthetised animals. The fact that inhibition from contralateral sites was abolished in spinalised animals shows that it is dependent on supraspinal structures. Our view is that noxious stimuli activate a widespread inhibition of reflex responses in the rest of the body. Diffuse noxious inhibitory controls (DNIC), by means of which nociceptive inputs inhibit responses to noxious stimuli in distant parts of the body, exert a widespread influence on spinal neurones via a supraspinally organised circuit (Villanueva & Le Bars, 1995) that could explain the inhibition of reflexes from off-limb sites in the present study. We cannot yet explain why mustard oil stimulation of some sites, notably those on the forelimbs, failed to generate any sort of change in hind limb reflexes. Repetitive, high intensity electrical stimulation of forelimb nerves inhibits MG reflexes (Taylor et al. 1991), so it may be simply that some sites require more intense activation than that provided by mustard oil in order to generate activity in the inhibitory system.

Sensitisation from deep tissues

In keeping with our previous observations (Clarke & Harris, 2001), noxious stimulation of deep structures was not particularly effective in generating central sensitisation in any preparation. This is in marked contrast to observations in rat, in which it was found that very long-lasting increases in withdrawal reflexes were obtained from noxious stimulation of the gastrocnemius muscle or the ankle joint (Wall & Woolf, 1984; Woolf & Wall, 1986). Studies in human subjects suggest that the hyperalgesia resulting from noxious stimulation of muscle is not particularly powerful or long-lasting (Babenko et al. 1999, 2000; Andersen et al. 2002).

Cardiovascular responses to mustard oil

Irrespective of the changes in reflexes generated by mustard oil stimuli, cardiovascular responses were almost homogeneous so long as the spinal cord was intact. We assume that the pressor responses to the stimulus were due to activation of brainstem defence areas such as the dorsolateral periaqueductal grey matter (Blessing, 1997). The relative uniformity of this response in each preparation indicates that mustard oil generated similar levels of nociceptive inflow wherever it was applied. We are currently testing this idea further by investigating patterns of fos evoked by application of the irritant to selected sites.


This study provides for the first time a complete description of the ‘sensitisation fields’ for reflexes to individual muscles. Central sensitisation of withdrawal reflexes is a spatially organised process centred on the area of skin withdrawn by the muscle involved in the reflex. For reflexes to flexor muscles, the sensitising area can expand to encompass the entire ipsilateral hind limb depending on the level of activity in descending systems. For the MG muscle, the sensitising area seems to be hard-wired into the spinal cord. Noxious stimuli applied off-limb can activate a rather non-selective inhibitory system that depends on a supraspinal circuit, reminiscent of DNIC. One aspect of spinal processing that has become clear from this study is the heterogeneity of controls to individual muscles, even where those muscles are activated from the same site. The level of tonic descending control, and the areas from which a reflex could be sensitised or depressed were different for every reflex studied. Thus, it would appear that observations made on a single spinal reflex (or other spinal events) cannot necessarily be applied to all spinal processing of nociceptive information. It would be very interesting to discover which aspects of the organisation of sensitisation of reflexes also apply to sensory transmission in the dorsal horn.

Supplementary Material

Supplementary Material (Quantitative analysis of the effects of mustard oil conditioning stimuli in decerebrate, decerebrate-spinal and anaesthetised rabbits)


This work was supported by the BBSRC. We would like to acknowledge the assistance of Laura Goulding in some of these experiments.

Supplementary material

The online version of this paper can be found at:


and contains supplementary material entitled:

Quantitative analysis of the effects of mustard oil conditioning stimuli in decerebrate, decerebrate-spinal and anaesthetised rabbits

These are tables that provide fully quantitative analyses of the effects of the mustard oil conditioning stimuli applied to different locations on the heel-medial gastrocnemius (MG), toes-tibialis anterior (TA) and toes-semitendinosus (ST) reflexes; the ongoing activity in the MG, TA and ST muscles; and arterial blood pressure in decerebrate, decerebrate-spinal and anaesthetised-intact rabbits. They should be read in conjunction with Figs 4--66 from the paper.


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