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Journal of Physiology (1991), 437, pp. 71-83 With 6 figures Printed in Great Britain
PROLONGED INHIBITION OF A SPINAL REFLEX AFTER INTENSE STIMULATION OF DISTANT PERIPHERAL NERVES IN THE DECEREBRATED RABBIT
BY J. S. TAYLOR*, R. I. NEALt, J. HARRIS, T. W. FORD AND R. W. CLARKE From the Department of Physiology and Environmental Science, University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough, Leicestershire LE12 5RD
(Received 2 August 1990) SUMMARY
1. In decerebrated rabbits, repetitive stimulation of the high-threshold afferents of the left common peroneal (CP) nerve evokes prolonged depression of the sural-gastrocnemius medialis (GM) reflex recorded in the same limb. This inhibition is antagonized by co-administration of the opioid antagonist naloxone with the a2adrenoceptor antagonist idazoxan. The present study was designed to investigate whether such inhibition could be elicited from the contralateral hindlimb or the forelimbs. 2. The sural-GM reflex of decerebrated rabbits was depressed for more than 15 min after stimulation of either ipsilateral or contralateral common peroneal (CP) or median nerves with 500 pulses of 20 V, 1 ms given at 5 Hz. The order of efficacy for generating this inhibition was ipsilateral CP > contralateral CP > ipsilateral median = contralateral median. In three of thirty-nine rabbits, stimulation of the median nerves caused facilitation of the sural-GM reflex. 3. Idazoxan (1-2 mg/kg i.v.) did not significantly alter the depressant effect of ipsilateral CP stimulation but reduced that evoked by either median nerve and almost abolished the inhibition evoked from the contralateral CP nerve. 4. Naloxone (0X25 mg/kg i.v.) reduced the effects of ipsilateral CP stimulation, did not alter the inhibition evoked from contralateral CP, and had equivocal actions on the responses to median nerve stimulation. 5. When given together, the two antagonists almost abolished the effects of stimulating the median nerves and the contralateral CP nerve, and markedly reduced the inhibition evoked from the ipsilateral CP nerve. 6. These data show that prolonged inhibition of the sural-GM reflex can be evoked by stimulation of nerves in all four limbs and that in each case the inhibition can be blocked or reduced by co-administration of antagonists to opioid and ac2-adrenergic * Present address: Department of Neuroscience, JHM Health Center, University of Florida, Gainesville, FL 32610, USA. t Present address: Department of Pharmacology, Merck, Sharp & Dohme, Neuroscience
Research Centre, MS 8698
Terlings Park, Eastwick Road, Harlow,
Essex.
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receptors. Such persistent inhibition of reflexes may serve to inhibit withdrawal reflexes in situations where interruptions to normal movement would be disadvantageous. INTRODUCTION
In decerebrated rabbits with a low thoracic spinal section, the reflex evoked by sural nerve stimulation in the ankle extensor gastrocnemius medialis (GM) is inhibited for 20-30 min after a brief period of repetitive stimulation of the fine axons of the common peroneal (CP) nerve (Catley, Clarke & Pascoe, 1984; Clarke, Ford & Taylor, 1989a). This inhibition is reversed by the opioid antagonist naloxone, which suggests that it is mediated by prolonged release of opioid peptides in the spinal cord. Stimulation of nerves other than CP does cause suppression of this extensor reflex, but only ipsilateral hindlimb nerves which terminate in spinal segments L7-S1 are effective (Catley et al. 1984). Noxious mechanical stimulation of the toes also results in prolonged, naloxone-reversible depression of GM reflex responses (Taylor, Pettit, Harris, Ford & Clarke, 1990), whereas noxious stimulation of the heel augments the response (Clarke, Ford, Harris & Taylor, 1990). It appears that the distribution of afferents which can evoke release of opioids into the sural-GM reflex pathway is limited to those innervating the most distal parts of the ipsilateral hindlimb. There are many reports of persistent spinal inhibition which can be activated by high-intensity stimuli, such as diffuse noxious inhibitory controls (DNIC; Le Bars, Dickenson & Besson, 1979), stress-induced analgesia (see Watkins & Mayer, 1982) and acupuncture-type analgesia (see Macdonald, 1989). These inhibitory phenomena can be activated from all over the body and can be reduced, but not blocked, by naloxone. Thus while it is probable that endogenous opioid peptides contribute to these processes, they are not the only mediators. Recently we reported that iterative stimulation of the CP nerve in decerebrated non-spinalized rabbits evoked a powerful depression of the sural-GM reflex which could only be substantially antagonized by co-administration of naloxone with the selective x2-adrenoceptor antagonist idazoxan (Clarke et al. 1989a). By analogy with the other forms of afferent-evoked inhibition described above, we surmised that, in non-spinalized rabbits, it would be possible to elicit suppression of the extensor reflex by stimulation of nerves distant to the ipsilateral hindlimb. Our hypothesis was that inhibition evoked from distant limbs would be primarily adrenergic, and the present experiments were designed to test these ideas. Some of the data reported here have appeared previously in abstracts (Neal & Taylor, 1989; Taylor, Neal, Clarke & Ford, 1989). METHODS
Experiments were performed on forty-eight male and female rabbits of New Zealand Red and White strains, weighing between 19 and 3-4 kg. Anaesthesia was induced by intravenous injection of methohexitone sodium (Brietal, Eli Lilly, 10 mg/kg initially) and maintained, after cannulation of the trachea, with halothane (2-4 %) in oxygen: nitrous oxide (30: 70). One carotid artery and one jugular vein were cannulated for recording arterial blood pressure and administration of drugs respectively. The second carotid artery was ligated. The head was clamped rigidly in a custom-built head holder using pointed ear bars, a mid-line craniotomy was performed and the rabbits were decerebrated by suction to the pre-collicular (twenty-seven animals) or mid-collicular (twenty-one) level.
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The left hindlimb was clamped rigidly at the ankle and the sural, GM and CP nerves were exposed through a dorsal incision. Each of these nerves was cut and placed over paired platinum electrodes for stimulation or recording. The GM nerve was crushed between the electrodes to give a monophasic signal. The contralateral CP nerve was exposed for stimulation by a similar dissection in twenty-one animals. The forelimbs were immobilized by fixing them to magnetic blocks and the median nerves exposed between the brachialis and triceps brachii muscles, which were deflected and sewn to brass rings to stabilize the limb. The left median nerve was exposed in twenty-seven rabbits and the right nerve in twenty-four. In each case the nerve was placed over twin platinum stimulating electrodes. When the limb nerve dissection was complete, anaesthesia was discontinued and the animals were paralysed with gallamine triethiodide (4 mg/kg i.v. initially) to be artificially ventilated on room air supplemented with oxygen. Arterial blood pressure was monitored and maintained so that mean pressure was > 50 mmHg. If the mean blood pressure decreased to less than 60 mmHg, intravenous injections of up to 10 ml of either 5 % Dglucose or 5 % dextran (MW 70000) in Ringer solution were given. If this manoeuvre did not have the desired effect, an infusion of adrenaline tartrate (20-60 ,ug/ml, given to effect) was started: the infusion itself had no effect on reflex responses, presumably because adrenaline does not cross the blood-brain barrier. The pump stroke was adjusted to give an end-tidal CO2 of 3-5 % (usually just around 4%) and core temperature was held between 37 5 and 38 5 °C by a thermostatic heating blanket. The sural nerve was stimulated with square-wave pulses of 01 ms duration and at intensities sufficient to produce a measurable reflex response in GM. The afferent volley was not recorded because there was insufficient space to insert the electrodes required to do this. Stimuli in the range of intensities employed (4-15 V) have been shown previously to recruit most if not all myelinated axons in the nerve (this would be in the range of 20-80 times threshold, see Clarke, Ford & Taylor, 1988). Twelve stimuli were applied at 1 Hz, repeated at intervals of 2 min. Reflex responses to these stimuli were recorded from the GM muscle nerve, the responses to the last eight stimuli averaged (to take account of 'wind-up' of the response, see Catley, Clarke & Pascoe, 1983), and the size of the response expressed as the voltage/time integral (area) of the muscle nerve neurogram. Only the area of the short-latency component of the response was measured (see Fig. 3). Conditioning stimuli were applied to the CP and median nerves as trains of 500 stimuli of 20 V and 1 ms, given at 5 Hz. Only one nerve was stimulated at a time and the order of application of conditioning stimuli, which were separated by intervals of at least 35 min, was determined by a blocked latin square. Conditioning stimuli were repeated in the presence of 0-25 mg/kg I.v. naloxone, a dose which has been shown previously to block the effects of CP nerve stimulation in spinalized rabbits (Clarke et al. 1989a). Where more than one nerve was stimulated in an animal, booster doses of 0-1 mg/kg were given between conditioning stimuli: the half-life of naloxone in this preparation is about 30 min (Catley et al. 1983). A separate group of rabbits received idazoxan (1-2 mg/kg i.v.). Doses in this range produce maximal facilitation of the sural-GM reflex in decerebrated rabbits (Clarke et al. 1988). Our experience with idazoxan suggests that it has a half-life rather longer than 1 h, and booster doses of 0-1-0-2 mg/kg were given between conditioning stimuli. The size of these doses was determined by trial and error. All animals which survived long enough then received the antagonist to which they had not already been exposed, i.e. those which had received naloxone were given idazoxan and vice versa. The drugs used in these experiments were idazoxan hydrochloride (a gift of Dr S. L. Dickinson, Reckitt & Colman) and naloxone hydrochloride (a gift of DuPont UK), which were dissolved in Ringer-Dale or 0-9 % NaCl solution to give an injection volume of 1 ml. All data were normalized and statistical comparisons were made with Student's t test (unpaired), assuming a significance level of 0 05. Some observations on the preparation Decerebrated, unanaesthetized rabbits are prone to sudden outbursts of sympathetic nervous activity which are characterized by rapid, short-lived (1-2 min) increases in blood pressure (up to 200 mmHg systolic) and heart rate, and which are usually presaged by bursts of activity in GM motoneurones. The sural-GM reflex is heavily inhibited for 1-4 min after such an episode. It was not always possible to relate these episodes to particular external stimuli. Attempts were made to reduce the frequency of these episodes and thus improve the stability of reflex responses. Animals prepared with a mid-collicular decerebration appeared to have a decreased likelihood of occurrence
J. S. TAYLOR AND OTHERS
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of these outbursts: they were observed in 8/21 of these animals as opposed to 15/27 of those decerebrated to the pre-collicular level. However, the effects of stimulation of the forelimb nerves were less repeatable in animals with mid-collicular section (see below). In later experiments, the sensory input to the preparation was minimized by applying local anaesthetic to all wounds and by packing small pledgets of cotton wool soaked with lignocaine into the external auditory meatus. Extraneous noises and lighting were reduced to a minimum, and troublesome episodes were encountered in 2/9 rabbits prepared in this way with pre-collicular decerebration. RESULTS
The effects of high-intensity stimulation of peripheral nerves in untreated rabbits Hindlimb nerves Repetitive stimulation of either ipsilateral CP (n = 38) or contralateral CP (n = 21) nerves always caused depression of the sural-GM reflex. The maximum effect was A 140 120 100
C,
E a)CDl
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Fig. 1. The effects on the sural-GM reflex of repetitive stimulation (500 pulses of 20 V, 1 ms given at 5 Hz) of: A, the ipsilateral (C], n = 26) and contralateral (U, n = 21) median nerves; and B, the ipsilateral (0, n 35) and contralateral (@, n 21) CP nerves. The stimuli were applied around time zero and each point is the mean + S.E. of the =
=
mean.
reached within 1 min of the end of the stimulus, at which time the reflex response had decreased to an average of 17 + 2 % (S.E.M.) of pre-stimulus levels after ipsilateral CP stimulation, and to 25 + 4 % after contralateral CP stimulation (Fig. IB). The reflex recovered to (i.e. it was not significantly different from) control values an average of
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Time (min relative to stimulus) Fig. 2. The effects of repetitive stimulation of the ipsilateral CP nerve in untreated animals (0, n = 35), after 1-2 mg/kg idazoxan (@, n = 21), after 0-25 mg/kg naloxone (OI, n = 12), and after co-administration of both drugs (U, n= 26). Each point is the mean + S.E. of the mean.
Untreated
After idazoxan
After idazoxan + naloxone
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Fig. 3. Recordings of GM responses to sural nerve stimulation from two rabbits, 1 min before and 1 min after stimulation of the contralateral common peroneal nerve with 500 shocks of 20 V, 1 ms, given at 5 Hz. The left-hand column shows responses in untreated animals; the central column after 1 mg/kg i.v. idazoxan (A, top frame) or 0-25 mg/kg i.v. naloxone (B, bottom frame); and the right-hand column after administration of both antagonists. The recordings are presented at different gains to allow comparison of relative changes in the size of the reflex after stimulation. The voltage scale is 50 ,uV at gain x 1; 100 gV at 2; 200 #uV at ÷ 4 and 400 ,uV at . 8. Each record is the average of eight sweeps, and the sural nerve was stimulated at the beginning of each sweep.
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19 min after the contralateral conditioning stimulus, but was still significantly less (P < 0025) than controls 31 min after the ipsilateral stimulus. The effects obtained from the ipsilateral CP nerve were significantly greater (P < 0-0005-0Y05) than those obtained from the contralateral CP at all times up to 25 min after the stimulus.
Forelimb nerves Iterative stimulation of the ipsilateral and contralateral median nerves depressed the sural-GM reflex in 26/29 and 21/22 rabbits respectively. Maximum depression was to a mean of 39+6% of pre-stimulus levels 1 min after ipsilateral median stimulation and to 38 + 6 % after activation of the contralateral nerve: recovery was complete within 21 and 19 min of stimulation respectively (Fig. 1A). The effects obtained from stimulation of the two median nerves were not significantly different from each other. Inhibition from the forelimb nerves was significantly less than that obtained from stimulation of the ipsilateral CP nerve at all times post-stimulus, but differed from the effects of contralateral CP stimulation only for the first minute (P < 005).
The effects of idazoxan and naloxone The selective ac2-receptor antagonist idazoxan increased the reflex as described previously (Clarke et al. 1988), to an average of 589 + 164 % (n = 26) of pre-drug levels when given intravenously in a dose of 1-2 mg/kg (see Fig. 3). The opioid antagonist naloxone (0-25 mg/kg i.v.) increased the sural-GM reflex to 216 + 55% (n = 22) of pre-drug levels: this was consistent with previous findings in decerebrated, non-spinalized rabbits (Clarke et al. 1988). When both drugs were given together, the reflex increased to an average of 1004 + 206 % (n = 40) of control levels.
Ipsilateral common peroneal stimulation The effects of conditioning stimuli applied to the ipsilateral CP nerve were not affected by idazoxan alone, but were significantly (P < 0025) reduced by naloxone (Fig. 2). One minute after ipsilateral CP stimulation in the presence of idazoxan the response was 21+4% (n = 21) of pre-stimulus levels, whereas the corresponding figure after naloxone was 37 + 7 % (n = 12). When both drugs were given together, the reflex decreased to 75 + 6 % (n = 26) of pre-stimulus levels after CP stimulation (Fig. 2), significantly (P < 0005) less inhibition than was seen in the presence of naloxone alone, but still statistically different from pre-stimulus values (P < 0-001). Contralateral common peroneal stimulation By contrast with the effects of ipsilateral CP stimulation, inhibition evoked from the contralateral CP was reduced by idazoxan (P < 0 005), but it was unaffected by naloxone pre-treatment (Figs 3 and 4). One minute after stimulation of this nerve in the presence of idazoxan the reflex was 75+ 8 % (n = 8) of pre-stimulus levels, but after naloxone the corresponding value was 26 + 9 % (n = 9). In the presence of naloxone, the effects of ipsilateral and contralateral CP stimulation were statistically indistinguishable. When both antagonists were present, contralateral CP stimulation resulted in a small but significant (P < 0 05) decrease in the sural-GM reflex only in the first minute after stimulation (Fig. 4).
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140 en
120
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0 4 8 12 16 20 24 28 32 Time (min relative to stimulus) Fig. 4. The effects of repetitive stimulation of the contralateral CP nerve in untreated animals (0, n = 21), after 1-2 mg/kg idazoxan (@, n = 8), after 0-25 mg/kg naloxone (E, n = 9), and after co-administration of both drugs (U, n = 13). Each point is the mean+ S.E. of the mean.
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Fig. 5. The effects of repetitive stimulation of the ipsilateral median nerve in untreated animals (0, n = 26), after 1-2 mg/kg idazoxan (@, n = 13), after 0-25 mg/kg naloxone (ED, n = 10), and after co-administration of both drugs (U, n = 17). Each point is the mean+ s.E. of the mean.
Stimulation of the median nerves Idazoxan significantly reduced the inhibition obtained from the ipsilateral (P < 0'01) and contralateral median (P < 0025) nerves (Figs 5 and 6), so that 1 min after stimulation the reflex was 66 + 8 (n = 13) and 65+12 % (n = 8) of pre-stimulus levels respectively. Forelimb nerve stimulation had mixed effects in the presence of naloxone. In most animals (9/10 with ipsilateral median nerve stimulation and 6/8
J. S. TAYLOR AND OTHERS with the contralateral nerve), the inhibition resulting from activation of forelimb afferents was unaffected. In the remaining rabbits, inhibition evoked from the median nerves was reversed to facilitation. Overall, in naloxone-treated rabbits the reflex decreased to 53 + 13 (n = 10) and 66 + 27 % (n = 8) of pre-stimulus levels after tetanic stimulation of the ipsilateral and contralateral median nerves respectively. These changes were not significantly different from those observed in untreated controls. 78
140 X5 120 E U,
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Fig. 6. The effects of repetitive stimulation of the contralateral median nerve in untreated animals (0, n = 21), after 1-2 mg/kg idazoxan (-, n = 8), after 025 mg/kg naloxone (Ii. n = 8). and after co-administration of both drugs (U, n = 14). Each point is the mean+ S.E. of the mean.
In the presence of the two antagonists, the inhibitory effects of stimulation of the median nerves were all but abolished (Figs 5 and 6), although there was a small, transient and statistically significant (P < 0 05) decrease in the reflex after activation of the contralateral median nerve.
Facilitation after stimulation of forelimb nerves Facilitation of the sural-GM reflex was seen after stimulation of the ipsilateral and contralateral median nerves in 3/29 and 1/22 rabbits respectively. Peak facilitation was to a mean of 289 + 61 % of pre-stimulus levels 1 min after ipsilateral median stimulation, and to 132 % after activation of contralateral afferents. This effect was observed only in animals decerebrated to the mid-collicular level. Where facilitation was obtained from stimulation of either ipsilateral or contralateral median nerves in untreated animals, it was abolished after idazoxan so that 1 min after ipsilateral and contralateral median nerve stimulation the reflex was 82 + 6 % (n = 3) and 102 % of pre-stimulus levels respectively. In the presence of idazoxan and naloxone together,
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ipsilateral median nerve stimulation increased the reflex response to 124 + 5 % (range) of pre-stimulus levels. Cardiovascular considerations The average mean arterial blood pressure in untreated rabbits was 99+3 mmHg (range 52-140 mmHg, n = 48): the corresponding figures after idazoxan alone, naloxone alone, and idazoxan with naloxone were 83+4 (range 51-120 mmHg, n = 26), 97+5 (range 50-130 mmHg, n = 22) and 79+4 mmHg (range 50-125 mmHg, n = 40) respectively, i.e. arterial pressure was significantly lower in idazoxan-treated rabbits. There was no correlation between arterial pressure and the absolute size of the reflex (correlation coefficient, r = 0087 in untreated animals) or the degree of inhibition obtained from limb nerve stimulation in these experiments: for stimulation of the ipsilateral CP nerve the correlation coefficient was 0 05 in untreated animals and 0 26 after administration of the two antagonists. Repetitive stimulation of either common peroneal or median nerves caused marked increases in blood pressure (mean increase 77 + 6 mmHg after ipsilateral CP stimulation) and heart rate which persisted for 1-5 min, and which were also observed, albeit somewhat reduced (to 58+7 mmHg for ipsilateral CP stimulation), in the presence of naloxone and idazoxan. Intravenous injection of adrenaline tartrate (10 ,ug/kg) produced increases in blood pressure of 88 + 9 mmHg (n = 5) of similar duration to those observed after nerve stimulation, but these were not accompanied by consistent changes in reflex responses. DISCUSSION
These experiments show that in decerebrated, non-spinalized rabbits long-lasting depression of the sural-GM reflex can be evoked by repetitive, high-intensity stimulation of nerves in any limb. This stimulus-evoked inhibition was always reduced or blocked by administration of idazoxan with naloxone, but the degree of inhibition observed and its sensitivity to adrenergic and/or opioid receptor blockade varied from limb to limb.
Ipsilateral hindlimb Iterative stimulation of the ipsilateral CP nerve generated the most profound and long-lasting inhibition of the sural-GM reflex. In a previous paper (Clarke et al. 1989a), it was argued that most of the effects of stimulating this nerve could be attributed to two independent mechanisms: (i) release of opioid peptides from spinal neurones; and (ii) liberation of noradrenaline (or adrenaline) from the terminals of descending fibres activated via a supraspinal pathway, to act at ox2-adrenoceptors in the spinal cord. These conclusions were based on the observations that CP-evoked inhibition of the sural-GM reflex is reversed by naloxone alone in spinalized rabbits (Catley et al. 1984; Clarke et al. 1989 a); that tonic descending inhibition of the reflex is blocked by intrathecal injection of idazoxan (Harris, Ford & Clarke, 1990); and that repetitive electrical stimulation of the sciatic nerves in the cat elicits release of
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noradrenaline from the surface of the spinal cord (Tyce & Yaksh, 1981). The present findings offer no reason to modify these conclusions, but it is necessary to point out that idazoxan is now known to bind with high affinity to a group of non-adrenergic imidazoline binding sites in some tissues in rabbit, including brain (e.g. Hamilton, Reid & Yakubu, 1988; Langin & Lafontan, 1989), so that results obtained with idazoxan must be treated with more circumspection than heretofore. Ipsilateral CP stimulation evoked inhibition even in the presence of idazoxan and naloxone. Assuming that the doses of the antagonists used were sufficient to cause complete blockade of opioid and ox2-receptors (see Methods), this observation suggests that at least one other transmitter system, as yet unidentified, is involved in generating inhibition after ipsilateral CP stimulation.
Contralateral hindlimb Inhibition obtained from the contralateral CP nerve was greatly reduced by idazoxan alone but was unaffected by naloxone, and therefore could be attributed mainly to the release of noradrenaline from descending fibres. This is consistent with the observation that repetitive stimulation of contralateral nerves failed to evoke naloxone-reversible suppression of the sural-GM reflex in spinalized rabbits (Catley et al. 1984), and supports the view that the naloxone-sensitive component of inhibition evoked from the ipsilateral nerve is largely the same as that seen in spinalized animals (Clarke et al. 1989a). Inhibition from forelimb nerve stimulation The effects of median nerve stimulation were much the same for each side of the body. In both cases the inhibition produced was significantly reduced by idazoxan alone but not, except in a small number of animals, by naloxone. However, complete blockade of the depressant action of these nerves was only achieved when both antagonists were given together, indicating, that, as for the ipsilateral hindlimb, opioidergic and adrenergic mechanisms were involved. Considering together the results of stimulating nerves in hind- and forelimbs, it appears that the neurones mediating prolonged, idazoxan-sensitive inhibition of the GM reflex response can be activated by inputs from all quarters of the body, and that the targets of their inhibitory actions are only poorly determined by the site from which they are activated. It has been suggested that many noradrenergic neurones in the pons project to the entire spinal cord (Westlund, Bowker, Ziegler & Coulter, 1982): such a diffuse pattern of projection could form the basis for the observations described in the present paper. The finding that endogenous opioids were involved in the effects initiated by stimulation of the median nerves was not anticipated, and may mean either that inputs from the forelimbs have access to opioidergic neurones in the lumbar segments via intraspinal descending fibres (cf. Cadden, Villanueva, Chitour & Le Bars, 1983), or that this component of the inhibition arises from supraspinal structures. Studies of forelimb nerve stimulation in rabbits with a cervical spinal section would help to answer this question.
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Facilitation from forelimb nerve stimulation That the sural-GM reflex was sometimes potentiated after median nerve stimulation in rabbits with mid-collicular decerebration, or after naloxone, shows that the central responses to intense afferent barrage are not fixed. Sectioning the brain stem at different levels can completely alter the patterns of reflexes obtained from stimulation of 'flexor reflex afferents' in the hindlimb of the cat (Holmqvist & Lundberg, 1961), so it is possible that forelimb afferents can activate descending systems which may be facilitatory and/or inhibitory to the sural-GM reflex and that a more caudal decerebration can favour activation of the former. The effects of idazoxan on this facilitation require further investigation.
Functional implications The sural-GM reflex pathway is probably involved in withdrawal of the limb from a noxious stimulus at the heel (Hagbarth, 1952; Clarke, Ford & Taylor, 1989b). Although no attempt was made in this study to identify the fibre groups responsible for generating the stimulus-evoked inhibition of GM reflex responses, previous experiments have shown that activation of small myelinated and/or non-myelinated axons is required to evoke inhibition from the ipsilateral CP nerve in both spinalized and non-spinalized animals (Clarke et al. 1989a). It would be surprising if this were not also true for nerves in the other limbs, and we believe that the inhibition described in the present paper would normally result from noxious stimulation of the limbs. The purpose of such stimulus-evoked inhibition would be to produce a widespread suppression of withdrawal reactions (which interrupt normal movement) at times when an animal is injured and under threat (see Duggan & Morton, 1988, for a full discussion of this idea). Furthermore, intense stimulation of distant peripheral nerves or tissues has been shown to suppress the responses of putative sensory transmission neurones in the dorsal horn of the rat (i.e. DNIC; Le Bars et al. 1979; Bouhassira, Le Bars & Villanueva, 1987), cat (Morton, Du, Xiao, Maisch & Zimmerman, 1988) and monkey (Chung, Fang, Hori, Lee & Willis, 1984; Chung, Lee, Hori, Endo & Willis, 1984), a phenomenon which is thought to underlie some forms of counter-irritation analgesia such as acupuncture (see Le Bars & Villanueva, 1988; Macdonald, 1989). There are many similarities between the stimulus-evoked depression of reflexes described in this paper and DNIC, and it is difficult to avoid the conclusion that they are manifestations of the same processes. Thus, in addition to modification of reflex responses, the inhibitory mechanisms described in this paper may have a role in modulating sensory inflow to the brain. Supported by the AFRC. J. S. T. and J. H. are SERC scholars. Our thanks to Graham Wood and Caroline Northway for helping in some of these experiments, to DuPont UK for the gift of naloxone and to Reckitt & Colman for the gift of idazoxan. REFERENCES
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CLARKE, R. W., FORD, T. W. & TAYLOR, J. S. (1989b). The reflex actions of selective stimulation of sural nerve C-fibres in the rabbit. Quarterly Journal of Experimental Physiology 74, 681-690. DUGGAN, A. W. & MORTON, C. R. (1988). Tonic descending inhibition and spinal nociceptive transmission. Progress in Brain Research 77. 193-211. HAGBARTH, K.-E. (1952). Excitatory and inhibitory skin areas for flexor and extensor motoneurones. Acta Physiologica Scandinavica
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HAMILTON, C. A., REID, J. L. & YAKUBU, M. A. (1988).[3H]-Yohimbine and [3H]-idazoxan bind to different sites in rabbit forebrain and kidney membranes. European Journal of Pharmacology 146, 345-349.
HARRIS, J., FORD, T. W. & CLARKE, R. XV. (1990). The effects of intrathecal application ofaadrenoceptor antagonists on the sural-gastrocnemius reflex in the decerebrate rabbit. Journal of Physiology 429, 130P.
HOLMQVIST, B. & LUNDBERG, A. (1961). Differential supraspinal control of synaptic actions evoked
by volleys in the flexion reflex afferents in alpha motoneurones. Acta Physiologica Scandinavica LANGIN, D. & LAFONTAN, M. (1989). [3H]-Idazoxan binding at non-a2-adrenoceptors in rabbit adipocyte membranes. European Journal of Pharmacology 159, 199-203. controls 54, suppl. 186, 1-51.
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on dorsal horn convergent neurones in the rat. Pain 6, 283-304. (DNIC). I.D. Effects LE L. & evidence for the activation
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descending controls by nociceptive afferent pathways. Progress in Brain Research 77, 275-299. MACDONALD, A. J. R. (1989). Acupuncture analgesia and therapy. In Textbook of Pain, ed. MELZACK, R. & WALL, P. D., pp. 906-919. Churchill Livingstone, London. MORTON, C. R., Du. H.-J., XIAO, H.-M., MAISCH, B. & ZIMMERMAN, M. (1988). Inhibition of nociceptive responses of dorsal horn neurones remote noxious afferent stimulation in the cat.
by NEAL, R. I. & TAYLOR, J. S. (1989). High intensity peripheral nerve stimulation evokes adrenergic and of a reflex in the decerebrated rabbit. Journal of Physiology Pain 34, 75-83.
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TAYLOR, J. S., NEAL, R. I., CLARKE, R. W. & FORD, T. WV. (1989). Suppression of a spinal reflex by
intense stimulation of near and remote peripheral nerves in the rabbit. Proceedings of the International Union of Physiological Sciences 17, 94. TAYLOR, J. S., PETTIT, J. S., HARRIS, J., FORD, T. WV. & CLARKE, R. W. (1990). Noxious stimulation of the toes evokes long-lasting, naloxone-reversible suppression of the suralgastrocnemius reflex in the rabbit. Brain Research 531, 263-268.
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TYCE, G. M. & YAKSH, T. L. (1981). Monoamine release from cat spinal cord by somatic stimuli: an intrinsic modulatory system. Journal of Physiology 314, 513-530. WATKINS, L. R. & MAYER, D. J. (1982). Organization of endogenous opiate and non-opiate pain
control systems. Science 216, 1185-1192. WESTLUND, K. N., BOWKER, R. M., ZIEGLER, M. G. & COULTER, J. D. (1982). Descending
noradrenergic projections and their spinal terminations. Progress in Brain Research 57, 219-238.
Journal of Physiology (1991), 437, pp. 71-83 With 6 figures Printed in Great Britain
PROLONGED INHIBITION OF A SPINAL REFLEX AFTER INTENSE STIMULATION OF DISTANT PERIPHERAL NERVES IN THE DECEREBRATED RABBIT
BY J. S. TAYLOR*, R. I. NEALt, J. HARRIS, T. W. FORD AND R. W. CLARKE From the Department of Physiology and Environmental Science, University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough, Leicestershire LE12 5RD
(Received 2 August 1990) SUMMARY
1. In decerebrated rabbits, repetitive stimulation of the high-threshold afferents of the left common peroneal (CP) nerve evokes prolonged depression of the sural-gastrocnemius medialis (GM) reflex recorded in the same limb. This inhibition is antagonized by co-administration of the opioid antagonist naloxone with the a2adrenoceptor antagonist idazoxan. The present study was designed to investigate whether such inhibition could be elicited from the contralateral hindlimb or the forelimbs. 2. The sural-GM reflex of decerebrated rabbits was depressed for more than 15 min after stimulation of either ipsilateral or contralateral common peroneal (CP) or median nerves with 500 pulses of 20 V, 1 ms given at 5 Hz. The order of efficacy for generating this inhibition was ipsilateral CP > contralateral CP > ipsilateral median = contralateral median. In three of thirty-nine rabbits, stimulation of the median nerves caused facilitation of the sural-GM reflex. 3. Idazoxan (1-2 mg/kg i.v.) did not significantly alter the depressant effect of ipsilateral CP stimulation but reduced that evoked by either median nerve and almost abolished the inhibition evoked from the contralateral CP nerve. 4. Naloxone (0X25 mg/kg i.v.) reduced the effects of ipsilateral CP stimulation, did not alter the inhibition evoked from contralateral CP, and had equivocal actions on the responses to median nerve stimulation. 5. When given together, the two antagonists almost abolished the effects of stimulating the median nerves and the contralateral CP nerve, and markedly reduced the inhibition evoked from the ipsilateral CP nerve. 6. These data show that prolonged inhibition of the sural-GM reflex can be evoked by stimulation of nerves in all four limbs and that in each case the inhibition can be blocked or reduced by co-administration of antagonists to opioid and ac2-adrenergic * Present address: Department of Neuroscience, JHM Health Center, University of Florida, Gainesville, FL 32610, USA. t Present address: Department of Pharmacology, Merck, Sharp & Dohme, Neuroscience
Research Centre, MS 8698
Terlings Park, Eastwick Road, Harlow,
Essex.
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receptors. Such persistent inhibition of reflexes may serve to inhibit withdrawal reflexes in situations where interruptions to normal movement would be disadvantageous. INTRODUCTION
In decerebrated rabbits with a low thoracic spinal section, the reflex evoked by sural nerve stimulation in the ankle extensor gastrocnemius medialis (GM) is inhibited for 20-30 min after a brief period of repetitive stimulation of the fine axons of the common peroneal (CP) nerve (Catley, Clarke & Pascoe, 1984; Clarke, Ford & Taylor, 1989a). This inhibition is reversed by the opioid antagonist naloxone, which suggests that it is mediated by prolonged release of opioid peptides in the spinal cord. Stimulation of nerves other than CP does cause suppression of this extensor reflex, but only ipsilateral hindlimb nerves which terminate in spinal segments L7-S1 are effective (Catley et al. 1984). Noxious mechanical stimulation of the toes also results in prolonged, naloxone-reversible depression of GM reflex responses (Taylor, Pettit, Harris, Ford & Clarke, 1990), whereas noxious stimulation of the heel augments the response (Clarke, Ford, Harris & Taylor, 1990). It appears that the distribution of afferents which can evoke release of opioids into the sural-GM reflex pathway is limited to those innervating the most distal parts of the ipsilateral hindlimb. There are many reports of persistent spinal inhibition which can be activated by high-intensity stimuli, such as diffuse noxious inhibitory controls (DNIC; Le Bars, Dickenson & Besson, 1979), stress-induced analgesia (see Watkins & Mayer, 1982) and acupuncture-type analgesia (see Macdonald, 1989). These inhibitory phenomena can be activated from all over the body and can be reduced, but not blocked, by naloxone. Thus while it is probable that endogenous opioid peptides contribute to these processes, they are not the only mediators. Recently we reported that iterative stimulation of the CP nerve in decerebrated non-spinalized rabbits evoked a powerful depression of the sural-GM reflex which could only be substantially antagonized by co-administration of naloxone with the selective x2-adrenoceptor antagonist idazoxan (Clarke et al. 1989a). By analogy with the other forms of afferent-evoked inhibition described above, we surmised that, in non-spinalized rabbits, it would be possible to elicit suppression of the extensor reflex by stimulation of nerves distant to the ipsilateral hindlimb. Our hypothesis was that inhibition evoked from distant limbs would be primarily adrenergic, and the present experiments were designed to test these ideas. Some of the data reported here have appeared previously in abstracts (Neal & Taylor, 1989; Taylor, Neal, Clarke & Ford, 1989). METHODS
Experiments were performed on forty-eight male and female rabbits of New Zealand Red and White strains, weighing between 19 and 3-4 kg. Anaesthesia was induced by intravenous injection of methohexitone sodium (Brietal, Eli Lilly, 10 mg/kg initially) and maintained, after cannulation of the trachea, with halothane (2-4 %) in oxygen: nitrous oxide (30: 70). One carotid artery and one jugular vein were cannulated for recording arterial blood pressure and administration of drugs respectively. The second carotid artery was ligated. The head was clamped rigidly in a custom-built head holder using pointed ear bars, a mid-line craniotomy was performed and the rabbits were decerebrated by suction to the pre-collicular (twenty-seven animals) or mid-collicular (twenty-one) level.
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The left hindlimb was clamped rigidly at the ankle and the sural, GM and CP nerves were exposed through a dorsal incision. Each of these nerves was cut and placed over paired platinum electrodes for stimulation or recording. The GM nerve was crushed between the electrodes to give a monophasic signal. The contralateral CP nerve was exposed for stimulation by a similar dissection in twenty-one animals. The forelimbs were immobilized by fixing them to magnetic blocks and the median nerves exposed between the brachialis and triceps brachii muscles, which were deflected and sewn to brass rings to stabilize the limb. The left median nerve was exposed in twenty-seven rabbits and the right nerve in twenty-four. In each case the nerve was placed over twin platinum stimulating electrodes. When the limb nerve dissection was complete, anaesthesia was discontinued and the animals were paralysed with gallamine triethiodide (4 mg/kg i.v. initially) to be artificially ventilated on room air supplemented with oxygen. Arterial blood pressure was monitored and maintained so that mean pressure was > 50 mmHg. If the mean blood pressure decreased to less than 60 mmHg, intravenous injections of up to 10 ml of either 5 % Dglucose or 5 % dextran (MW 70000) in Ringer solution were given. If this manoeuvre did not have the desired effect, an infusion of adrenaline tartrate (20-60 ,ug/ml, given to effect) was started: the infusion itself had no effect on reflex responses, presumably because adrenaline does not cross the blood-brain barrier. The pump stroke was adjusted to give an end-tidal CO2 of 3-5 % (usually just around 4%) and core temperature was held between 37 5 and 38 5 °C by a thermostatic heating blanket. The sural nerve was stimulated with square-wave pulses of 01 ms duration and at intensities sufficient to produce a measurable reflex response in GM. The afferent volley was not recorded because there was insufficient space to insert the electrodes required to do this. Stimuli in the range of intensities employed (4-15 V) have been shown previously to recruit most if not all myelinated axons in the nerve (this would be in the range of 20-80 times threshold, see Clarke, Ford & Taylor, 1988). Twelve stimuli were applied at 1 Hz, repeated at intervals of 2 min. Reflex responses to these stimuli were recorded from the GM muscle nerve, the responses to the last eight stimuli averaged (to take account of 'wind-up' of the response, see Catley, Clarke & Pascoe, 1983), and the size of the response expressed as the voltage/time integral (area) of the muscle nerve neurogram. Only the area of the short-latency component of the response was measured (see Fig. 3). Conditioning stimuli were applied to the CP and median nerves as trains of 500 stimuli of 20 V and 1 ms, given at 5 Hz. Only one nerve was stimulated at a time and the order of application of conditioning stimuli, which were separated by intervals of at least 35 min, was determined by a blocked latin square. Conditioning stimuli were repeated in the presence of 0-25 mg/kg I.v. naloxone, a dose which has been shown previously to block the effects of CP nerve stimulation in spinalized rabbits (Clarke et al. 1989a). Where more than one nerve was stimulated in an animal, booster doses of 0-1 mg/kg were given between conditioning stimuli: the half-life of naloxone in this preparation is about 30 min (Catley et al. 1983). A separate group of rabbits received idazoxan (1-2 mg/kg i.v.). Doses in this range produce maximal facilitation of the sural-GM reflex in decerebrated rabbits (Clarke et al. 1988). Our experience with idazoxan suggests that it has a half-life rather longer than 1 h, and booster doses of 0-1-0-2 mg/kg were given between conditioning stimuli. The size of these doses was determined by trial and error. All animals which survived long enough then received the antagonist to which they had not already been exposed, i.e. those which had received naloxone were given idazoxan and vice versa. The drugs used in these experiments were idazoxan hydrochloride (a gift of Dr S. L. Dickinson, Reckitt & Colman) and naloxone hydrochloride (a gift of DuPont UK), which were dissolved in Ringer-Dale or 0-9 % NaCl solution to give an injection volume of 1 ml. All data were normalized and statistical comparisons were made with Student's t test (unpaired), assuming a significance level of 0 05. Some observations on the preparation Decerebrated, unanaesthetized rabbits are prone to sudden outbursts of sympathetic nervous activity which are characterized by rapid, short-lived (1-2 min) increases in blood pressure (up to 200 mmHg systolic) and heart rate, and which are usually presaged by bursts of activity in GM motoneurones. The sural-GM reflex is heavily inhibited for 1-4 min after such an episode. It was not always possible to relate these episodes to particular external stimuli. Attempts were made to reduce the frequency of these episodes and thus improve the stability of reflex responses. Animals prepared with a mid-collicular decerebration appeared to have a decreased likelihood of occurrence
J. S. TAYLOR AND OTHERS
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of these outbursts: they were observed in 8/21 of these animals as opposed to 15/27 of those decerebrated to the pre-collicular level. However, the effects of stimulation of the forelimb nerves were less repeatable in animals with mid-collicular section (see below). In later experiments, the sensory input to the preparation was minimized by applying local anaesthetic to all wounds and by packing small pledgets of cotton wool soaked with lignocaine into the external auditory meatus. Extraneous noises and lighting were reduced to a minimum, and troublesome episodes were encountered in 2/9 rabbits prepared in this way with pre-collicular decerebration. RESULTS
The effects of high-intensity stimulation of peripheral nerves in untreated rabbits Hindlimb nerves Repetitive stimulation of either ipsilateral CP (n = 38) or contralateral CP (n = 21) nerves always caused depression of the sural-GM reflex. The maximum effect was A 140 120 100
C,
E a)CDl
CD E x 4-
x 0 -) a) 0 CD
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Fig. 1. The effects on the sural-GM reflex of repetitive stimulation (500 pulses of 20 V, 1 ms given at 5 Hz) of: A, the ipsilateral (C], n = 26) and contralateral (U, n = 21) median nerves; and B, the ipsilateral (0, n 35) and contralateral (@, n 21) CP nerves. The stimuli were applied around time zero and each point is the mean + S.E. of the =
=
mean.
reached within 1 min of the end of the stimulus, at which time the reflex response had decreased to an average of 17 + 2 % (S.E.M.) of pre-stimulus levels after ipsilateral CP stimulation, and to 25 + 4 % after contralateral CP stimulation (Fig. IB). The reflex recovered to (i.e. it was not significantly different from) control values an average of
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140 cn
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Time (min relative to stimulus) Fig. 2. The effects of repetitive stimulation of the ipsilateral CP nerve in untreated animals (0, n = 35), after 1-2 mg/kg idazoxan (@, n = 21), after 0-25 mg/kg naloxone (OI, n = 12), and after co-administration of both drugs (U, n= 26). Each point is the mean + S.E. of the mean.
Untreated
After idazoxan
After idazoxan + naloxone
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Fig. 3. Recordings of GM responses to sural nerve stimulation from two rabbits, 1 min before and 1 min after stimulation of the contralateral common peroneal nerve with 500 shocks of 20 V, 1 ms, given at 5 Hz. The left-hand column shows responses in untreated animals; the central column after 1 mg/kg i.v. idazoxan (A, top frame) or 0-25 mg/kg i.v. naloxone (B, bottom frame); and the right-hand column after administration of both antagonists. The recordings are presented at different gains to allow comparison of relative changes in the size of the reflex after stimulation. The voltage scale is 50 ,uV at gain x 1; 100 gV at 2; 200 #uV at ÷ 4 and 400 ,uV at . 8. Each record is the average of eight sweeps, and the sural nerve was stimulated at the beginning of each sweep.
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19 min after the contralateral conditioning stimulus, but was still significantly less (P < 0025) than controls 31 min after the ipsilateral stimulus. The effects obtained from the ipsilateral CP nerve were significantly greater (P < 0-0005-0Y05) than those obtained from the contralateral CP at all times up to 25 min after the stimulus.
Forelimb nerves Iterative stimulation of the ipsilateral and contralateral median nerves depressed the sural-GM reflex in 26/29 and 21/22 rabbits respectively. Maximum depression was to a mean of 39+6% of pre-stimulus levels 1 min after ipsilateral median stimulation and to 38 + 6 % after activation of the contralateral nerve: recovery was complete within 21 and 19 min of stimulation respectively (Fig. 1A). The effects obtained from stimulation of the two median nerves were not significantly different from each other. Inhibition from the forelimb nerves was significantly less than that obtained from stimulation of the ipsilateral CP nerve at all times post-stimulus, but differed from the effects of contralateral CP stimulation only for the first minute (P < 005).
The effects of idazoxan and naloxone The selective ac2-receptor antagonist idazoxan increased the reflex as described previously (Clarke et al. 1988), to an average of 589 + 164 % (n = 26) of pre-drug levels when given intravenously in a dose of 1-2 mg/kg (see Fig. 3). The opioid antagonist naloxone (0-25 mg/kg i.v.) increased the sural-GM reflex to 216 + 55% (n = 22) of pre-drug levels: this was consistent with previous findings in decerebrated, non-spinalized rabbits (Clarke et al. 1988). When both drugs were given together, the reflex increased to an average of 1004 + 206 % (n = 40) of control levels.
Ipsilateral common peroneal stimulation The effects of conditioning stimuli applied to the ipsilateral CP nerve were not affected by idazoxan alone, but were significantly (P < 0025) reduced by naloxone (Fig. 2). One minute after ipsilateral CP stimulation in the presence of idazoxan the response was 21+4% (n = 21) of pre-stimulus levels, whereas the corresponding figure after naloxone was 37 + 7 % (n = 12). When both drugs were given together, the reflex decreased to 75 + 6 % (n = 26) of pre-stimulus levels after CP stimulation (Fig. 2), significantly (P < 0005) less inhibition than was seen in the presence of naloxone alone, but still statistically different from pre-stimulus values (P < 0-001). Contralateral common peroneal stimulation By contrast with the effects of ipsilateral CP stimulation, inhibition evoked from the contralateral CP was reduced by idazoxan (P < 0 005), but it was unaffected by naloxone pre-treatment (Figs 3 and 4). One minute after stimulation of this nerve in the presence of idazoxan the reflex was 75+ 8 % (n = 8) of pre-stimulus levels, but after naloxone the corresponding value was 26 + 9 % (n = 9). In the presence of naloxone, the effects of ipsilateral and contralateral CP stimulation were statistically indistinguishable. When both antagonists were present, contralateral CP stimulation resulted in a small but significant (P < 0 05) decrease in the sural-GM reflex only in the first minute after stimulation (Fig. 4).
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140 en
120
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100 CL
a) E o-0 60X '
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0 4 8 12 16 20 24 28 32 Time (min relative to stimulus) Fig. 4. The effects of repetitive stimulation of the contralateral CP nerve in untreated animals (0, n = 21), after 1-2 mg/kg idazoxan (@, n = 8), after 0-25 mg/kg naloxone (E, n = 9), and after co-administration of both drugs (U, n = 13). Each point is the mean+ S.E. of the mean.
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Fig. 5. The effects of repetitive stimulation of the ipsilateral median nerve in untreated animals (0, n = 26), after 1-2 mg/kg idazoxan (@, n = 13), after 0-25 mg/kg naloxone (ED, n = 10), and after co-administration of both drugs (U, n = 17). Each point is the mean+ s.E. of the mean.
Stimulation of the median nerves Idazoxan significantly reduced the inhibition obtained from the ipsilateral (P < 0'01) and contralateral median (P < 0025) nerves (Figs 5 and 6), so that 1 min after stimulation the reflex was 66 + 8 (n = 13) and 65+12 % (n = 8) of pre-stimulus levels respectively. Forelimb nerve stimulation had mixed effects in the presence of naloxone. In most animals (9/10 with ipsilateral median nerve stimulation and 6/8
J. S. TAYLOR AND OTHERS with the contralateral nerve), the inhibition resulting from activation of forelimb afferents was unaffected. In the remaining rabbits, inhibition evoked from the median nerves was reversed to facilitation. Overall, in naloxone-treated rabbits the reflex decreased to 53 + 13 (n = 10) and 66 + 27 % (n = 8) of pre-stimulus levels after tetanic stimulation of the ipsilateral and contralateral median nerves respectively. These changes were not significantly different from those observed in untreated controls. 78
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Fig. 6. The effects of repetitive stimulation of the contralateral median nerve in untreated animals (0, n = 21), after 1-2 mg/kg idazoxan (-, n = 8), after 025 mg/kg naloxone (Ii. n = 8). and after co-administration of both drugs (U, n = 14). Each point is the mean+ S.E. of the mean.
In the presence of the two antagonists, the inhibitory effects of stimulation of the median nerves were all but abolished (Figs 5 and 6), although there was a small, transient and statistically significant (P < 0 05) decrease in the reflex after activation of the contralateral median nerve.
Facilitation after stimulation of forelimb nerves Facilitation of the sural-GM reflex was seen after stimulation of the ipsilateral and contralateral median nerves in 3/29 and 1/22 rabbits respectively. Peak facilitation was to a mean of 289 + 61 % of pre-stimulus levels 1 min after ipsilateral median stimulation, and to 132 % after activation of contralateral afferents. This effect was observed only in animals decerebrated to the mid-collicular level. Where facilitation was obtained from stimulation of either ipsilateral or contralateral median nerves in untreated animals, it was abolished after idazoxan so that 1 min after ipsilateral and contralateral median nerve stimulation the reflex was 82 + 6 % (n = 3) and 102 % of pre-stimulus levels respectively. In the presence of idazoxan and naloxone together,
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ipsilateral median nerve stimulation increased the reflex response to 124 + 5 % (range) of pre-stimulus levels. Cardiovascular considerations The average mean arterial blood pressure in untreated rabbits was 99+3 mmHg (range 52-140 mmHg, n = 48): the corresponding figures after idazoxan alone, naloxone alone, and idazoxan with naloxone were 83+4 (range 51-120 mmHg, n = 26), 97+5 (range 50-130 mmHg, n = 22) and 79+4 mmHg (range 50-125 mmHg, n = 40) respectively, i.e. arterial pressure was significantly lower in idazoxan-treated rabbits. There was no correlation between arterial pressure and the absolute size of the reflex (correlation coefficient, r = 0087 in untreated animals) or the degree of inhibition obtained from limb nerve stimulation in these experiments: for stimulation of the ipsilateral CP nerve the correlation coefficient was 0 05 in untreated animals and 0 26 after administration of the two antagonists. Repetitive stimulation of either common peroneal or median nerves caused marked increases in blood pressure (mean increase 77 + 6 mmHg after ipsilateral CP stimulation) and heart rate which persisted for 1-5 min, and which were also observed, albeit somewhat reduced (to 58+7 mmHg for ipsilateral CP stimulation), in the presence of naloxone and idazoxan. Intravenous injection of adrenaline tartrate (10 ,ug/kg) produced increases in blood pressure of 88 + 9 mmHg (n = 5) of similar duration to those observed after nerve stimulation, but these were not accompanied by consistent changes in reflex responses. DISCUSSION
These experiments show that in decerebrated, non-spinalized rabbits long-lasting depression of the sural-GM reflex can be evoked by repetitive, high-intensity stimulation of nerves in any limb. This stimulus-evoked inhibition was always reduced or blocked by administration of idazoxan with naloxone, but the degree of inhibition observed and its sensitivity to adrenergic and/or opioid receptor blockade varied from limb to limb.
Ipsilateral hindlimb Iterative stimulation of the ipsilateral CP nerve generated the most profound and long-lasting inhibition of the sural-GM reflex. In a previous paper (Clarke et al. 1989a), it was argued that most of the effects of stimulating this nerve could be attributed to two independent mechanisms: (i) release of opioid peptides from spinal neurones; and (ii) liberation of noradrenaline (or adrenaline) from the terminals of descending fibres activated via a supraspinal pathway, to act at ox2-adrenoceptors in the spinal cord. These conclusions were based on the observations that CP-evoked inhibition of the sural-GM reflex is reversed by naloxone alone in spinalized rabbits (Catley et al. 1984; Clarke et al. 1989 a); that tonic descending inhibition of the reflex is blocked by intrathecal injection of idazoxan (Harris, Ford & Clarke, 1990); and that repetitive electrical stimulation of the sciatic nerves in the cat elicits release of
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noradrenaline from the surface of the spinal cord (Tyce & Yaksh, 1981). The present findings offer no reason to modify these conclusions, but it is necessary to point out that idazoxan is now known to bind with high affinity to a group of non-adrenergic imidazoline binding sites in some tissues in rabbit, including brain (e.g. Hamilton, Reid & Yakubu, 1988; Langin & Lafontan, 1989), so that results obtained with idazoxan must be treated with more circumspection than heretofore. Ipsilateral CP stimulation evoked inhibition even in the presence of idazoxan and naloxone. Assuming that the doses of the antagonists used were sufficient to cause complete blockade of opioid and ox2-receptors (see Methods), this observation suggests that at least one other transmitter system, as yet unidentified, is involved in generating inhibition after ipsilateral CP stimulation.
Contralateral hindlimb Inhibition obtained from the contralateral CP nerve was greatly reduced by idazoxan alone but was unaffected by naloxone, and therefore could be attributed mainly to the release of noradrenaline from descending fibres. This is consistent with the observation that repetitive stimulation of contralateral nerves failed to evoke naloxone-reversible suppression of the sural-GM reflex in spinalized rabbits (Catley et al. 1984), and supports the view that the naloxone-sensitive component of inhibition evoked from the ipsilateral nerve is largely the same as that seen in spinalized animals (Clarke et al. 1989a). Inhibition from forelimb nerve stimulation The effects of median nerve stimulation were much the same for each side of the body. In both cases the inhibition produced was significantly reduced by idazoxan alone but not, except in a small number of animals, by naloxone. However, complete blockade of the depressant action of these nerves was only achieved when both antagonists were given together, indicating, that, as for the ipsilateral hindlimb, opioidergic and adrenergic mechanisms were involved. Considering together the results of stimulating nerves in hind- and forelimbs, it appears that the neurones mediating prolonged, idazoxan-sensitive inhibition of the GM reflex response can be activated by inputs from all quarters of the body, and that the targets of their inhibitory actions are only poorly determined by the site from which they are activated. It has been suggested that many noradrenergic neurones in the pons project to the entire spinal cord (Westlund, Bowker, Ziegler & Coulter, 1982): such a diffuse pattern of projection could form the basis for the observations described in the present paper. The finding that endogenous opioids were involved in the effects initiated by stimulation of the median nerves was not anticipated, and may mean either that inputs from the forelimbs have access to opioidergic neurones in the lumbar segments via intraspinal descending fibres (cf. Cadden, Villanueva, Chitour & Le Bars, 1983), or that this component of the inhibition arises from supraspinal structures. Studies of forelimb nerve stimulation in rabbits with a cervical spinal section would help to answer this question.
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Facilitation from forelimb nerve stimulation That the sural-GM reflex was sometimes potentiated after median nerve stimulation in rabbits with mid-collicular decerebration, or after naloxone, shows that the central responses to intense afferent barrage are not fixed. Sectioning the brain stem at different levels can completely alter the patterns of reflexes obtained from stimulation of 'flexor reflex afferents' in the hindlimb of the cat (Holmqvist & Lundberg, 1961), so it is possible that forelimb afferents can activate descending systems which may be facilitatory and/or inhibitory to the sural-GM reflex and that a more caudal decerebration can favour activation of the former. The effects of idazoxan on this facilitation require further investigation.
Functional implications The sural-GM reflex pathway is probably involved in withdrawal of the limb from a noxious stimulus at the heel (Hagbarth, 1952; Clarke, Ford & Taylor, 1989b). Although no attempt was made in this study to identify the fibre groups responsible for generating the stimulus-evoked inhibition of GM reflex responses, previous experiments have shown that activation of small myelinated and/or non-myelinated axons is required to evoke inhibition from the ipsilateral CP nerve in both spinalized and non-spinalized animals (Clarke et al. 1989a). It would be surprising if this were not also true for nerves in the other limbs, and we believe that the inhibition described in the present paper would normally result from noxious stimulation of the limbs. The purpose of such stimulus-evoked inhibition would be to produce a widespread suppression of withdrawal reactions (which interrupt normal movement) at times when an animal is injured and under threat (see Duggan & Morton, 1988, for a full discussion of this idea). Furthermore, intense stimulation of distant peripheral nerves or tissues has been shown to suppress the responses of putative sensory transmission neurones in the dorsal horn of the rat (i.e. DNIC; Le Bars et al. 1979; Bouhassira, Le Bars & Villanueva, 1987), cat (Morton, Du, Xiao, Maisch & Zimmerman, 1988) and monkey (Chung, Fang, Hori, Lee & Willis, 1984; Chung, Lee, Hori, Endo & Willis, 1984), a phenomenon which is thought to underlie some forms of counter-irritation analgesia such as acupuncture (see Le Bars & Villanueva, 1988; Macdonald, 1989). There are many similarities between the stimulus-evoked depression of reflexes described in this paper and DNIC, and it is difficult to avoid the conclusion that they are manifestations of the same processes. Thus, in addition to modification of reflex responses, the inhibitory mechanisms described in this paper may have a role in modulating sensory inflow to the brain. Supported by the AFRC. J. S. T. and J. H. are SERC scholars. Our thanks to Graham Wood and Caroline Northway for helping in some of these experiments, to DuPont UK for the gift of naloxone and to Reckitt & Colman for the gift of idazoxan. REFERENCES
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