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The antinociceptive effects of anterior pretectal stimulation in tests using thermal, mechanical and chemical noxious stimuli
195
Pain, 44 (1991) 195-200 0 1991 Elsevier Science Publishers B.V. 0304-3959/91/%03.50 ADONIS
030439599100077M
PAIN 01731
The antinociceptive effects of anterior pretectal stimulation in tests using thermal, mechanical and chemical noxious stimuli D.G. Wilson, H. Rees and M.H.T. Roberts Depamnem
of Physk&gy,
Uniuersity of Wales College of Cardi& Cardiff CFI ISS (U.K.)
(Received 19 March 1990, revision received 29 June 1990, accepted 14 August 1990)
Summary
Four behavioural tests have been used to study the antinociceptive effects of electrical stimulation of the anterior pretectal nucleus (APtN) in the rat. The antinociceptive effects of stimulating this nucleus, which lies dorsally in the posterior diencephalon, have recently been studied extensively but always using briefly applied heat stimuli. It is reported here that APtN stimulation effectively inhibited responses to briefly applied noxious pressure and longer-lasting noxious chemical (formalin) stimuli. Although the tail-flick reflex to noxious heat was very potently depressed by APtN stimulation, responses to noxious heat in the hot-plate test were not. Three doses of morphine were also studied with each test and it was concluded that 15 set of 35 PIA r.m.s. current into the APtN was as effective as 3-5 mg/kg morphine S.C.in the rat. Key words: Pain; Antinociception;
Paw pressure test; Formalin test; Hot plate; Tail flick
Stimulation of the anterior pretectal nucleus (APtN) has been shown to inhibit potently the tail-flick reflex to noxious heat without disrupting motor behaviour or responses to non-noxious stimuli fl5]. The stimulation is not aversive. The APtN is an import~t relay of the antinociceptive effects of stimulating the somatosensory cortex [2], the dorsal columns [13] and deep mesencephalic nuclei [6]. Spinal lamina V cell responses to noxious thermal stimuli are inhibited by APtN stimulation. The descending inhibitory pathway runs in the dorsolateral funiculus. All these data have been obtained using briefly applied thermal stimuli and the behavioural data have all been obtained with studies of the spinal reflex ‘tail-flick’ response. The tail flick does not require the integrative actions of higher centres [7] and its sole use as a means of identifying antinociception has been questioned [l&19]. We have therefore studied the effects of electrical stimulation of the APtN
Corres~omfenee to: Dr. H. Rees, Department of Physjology, University of Wales College of Cardiff, PO Box 902, Cardiff CFl lSS, U.K.
on behaviours which are thought to be mediated supraspinally. Furthermore, antinociceptive tests employing brief stimuli which give sensations of sharp pain to human volunteers may be affected differently by analgesics from those tests which give sensations of prolonged burning pain. The effects of APtN stimulation on both types of test have been studied. The antinociceptive tests which have been employed are: (1) the hot-plate test, (2) the paw pressure test, and (3) the formalin test. In addition, the sensitivities of these tests to APtN stimulation have been compared to the tail-flick test. For the purpose of comparison the sensitivities of the tests, as used in this laboratory, to varying doses of morphine have also been investigated.
Methods and experimental
design
One hundred male Wistar rats (270-290 g) were used. The animals were divided into two groups, one group was used in studies which involved electrical stimulation of the APtN and the other to compare the effects of morphine. Sixty-four rats were used in the study of the effects of morphine, they were divided into 4 groups of 16,
each group being used to study responses to a different analgesiometric test. The animals were tested on 2 separate occasions separated by at least 1 week. Animals received a single subcutaneous injection of either 1.5, 3, or 5 mg/ kg morphine or 0.05 ml saline. On the second test the animal received a different dose of morphine or saline. Procedures for the implantation of guide cannulae, the tail-flick test and for electrical stimulation of the APtN have been described in details elsewhere [15]. Briefly, 12 mm lengths of hypoder~c needle tubing were stereotaxically positioned into a hole drilled through the skull and held in place by dental acrylic. The end of the cannula lay 3 mm above the APtN. A unipolar insulated steel wire was cut to 15 mm and inserted into the cannula so that the tip lay in the APtN. Fifty Hz sine wave current of 35 PA r.m.s. was applied to the electrode for 15 sec. Four analgesiometric tests were used. The tail-flick procedure has been described elsewhere [15]. Briefly, the rat was placed into a clear glass tube from which the tail protruded. A part of the tail between 2 and 6 cm from the tip was placed onto a nichrome wire coil at room temperature. A calibrated current was passed through the coil so as to raise its temperature 9” C/set. After about 3 set (coil temperature about 45’C) normal rats flicked the tail away from the heat source. If no response occurred the tail was removed after 6 set to prevent tissue damage. Scores were normalised according to the following formula: Index of analgesia =
latency - control latency 6 - control latency
The control latency was the average of 3 responses immediately preceding the injection or APtN stimulation. The paw pressure test used the ‘Ugo Basile Analgesy-meter.’ Each rat was gently held with a hind paw placed under a pressure pad. The force applied to the paw was progressively increased, at a rate of 48 g/set to a maximum weight of 750 g or until either a vocalisation or struggle was evoked. The pressure was immediately removed and the force required to elicit the end-point response was noted. Data were normal&d in the same way as described for the tail-flick test, except that the pressure applied to achieve the end-point was used as the score. Baseline levels were also determined as before. In the formalin test the general methods outlined by Cohen and Melzack [3] have been used with a few minor alterations. The test involved the use of clear glass or perspex observation chambers with mirrors placed at an angle underneath them. The behaviour of the rats was examined and scored for 30 set periods at 5 min intervals both before and after the intradermal injection of 0.05 ml of a 2.5% formalin solution into the
ptantar surface of the right hind paw. The score was based on the position and utilisation of the right hind paw. Normal weight-bearing behaviour was assigned a score of ‘0.’ whilst the shaking or licking of the paw was assigned a ‘3.’ Scores of ‘1’ and ‘2’ represent a gradation between the two extremes. The scores were fed into an on-line computer, which was used in the analysis of the data. Intradermal formalin caused an increase in behavioural scores at 5 min followed by a rapid decline to just above normal score levels 5 min later. This was foBowed by a second rise in scores which was sustained for 20 min before gradually declining. This response was consistent for all control animals and is in agreement with other studies using this analgesiometric test [3,4]. The experimental variable was applied prior to the second increase in behavioural score. The first increase was too variable to allow meaningful comparisons, slight differences in time between formalin injection and recording periods affect the magnitude of the recorded peak giving variable rest&s. In contrast. the second peak in behavioural score provides a prolonged steady plateau response and this response has been taken by others to represent tonic pain [1,5]. In the hot-plate test the general methods outlined by others have been followed [11,16]. The hot-plate test comprised a high-sided enamel box held semi-submerged in a heated water bath. The water temperature was thermostatically controlled so that the enamel surface was at 55°C. The animal was placed on to the plate and the period of time which elapsed until a hind paw was licked was recorded. The paw lick was taken to be the end-point of the test, and the rat was immediately removed from the box. If the animal did not lick the hind paw within the 30 set test period it was removed from the hot plate to prevent tissue damage. Determinations of statistical significance were done using the non-parametric Mann-Whitney U test.
Results
Behavioural tests conducted in different laboratories or with different strains of rats vary greatly with respect to the effectiveness of analgesic treatments. Standard doses of morphine have been administered in order to define the sensitivity of these tests to this well known analgesic and to provide a baseline for comparison of the effects of APtN stimulation. Effects of subcutaneous morphine Subcutaneously administered morphine at 5, 3 and 1.5 mg/kg caused a rapid elevation of tail-flick latency to maximal or near maximal levels. The effects were shorter lasting for the lower doses but, as illustrated by Fig. lA, complete recovery of baseline latencies did not occur for any dose within 90 min of the injection.
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Fig. 1. A: the effectiveness of 3 doses of morphine in causing inhibition of the tail-flick reflex to noxious heat. Calculation of the index of analgesia is described in the text. Subcutaneous injections of morphine 5 mg/kg (A), 3 mg/kg (+) or 1.5 mg/kg (0) or saline (A) were made just prior to time 0. Each group contained 8 animals. All doses of morphine were effective but the lower doses acted for a shorter period. Saline was without effect. B: the antinociceptive effect of subcutaneous morphine in the paw pressure test. Each group contained 8 animals. All doses of morphine increased the vocalisation latency. The increase was gradual in onset, maximal effects being observed 20-50 min post injection. The greatest increase was seen following an administration of 5 mg/kg morphine (A). There was little difference between the (+) and 1.5 (0) mg/kg doses. No increase was seen with saline-treated animals. C: the antinociceptive potency of morphine in the hot-plate test. Each group contained 8 animals. 5 (A), 3 (+) and 1.5 (0) mg/kg morphine all increased the paw lick latency. Mine (A) had no effect. The large standard error bars indicate the considerable variability of the weak antinociceptive effect of morphine. * Significant difference from control values (P i 0.05).
The paw pressure test was less sensitive to the effects of morphine and the variability was much greater. The data are shown in Fig. 1B. Maximal scores were not achieved except for one point with the highest dose. The onset of the effects of morphine was much slower than with the tail-flick test and peak effects were seen 50 min after the injection. The duration of the effect of morphine seemed to be less and baseline levels were nearly recovered 90 min after the injection. The greatest effect was seen with the higher dose but the test did not discriminate between the two lower doses of morphine. The results obtained with the hot-plate test (illustrated in Fig. 1C) show even greater variability than the paw pressure test. The hot-plate test is not usually used to display the full time course of an analgesic effect and it is clear from the data why this is so. Nevertheless, a clear inhibition of the response latency occurred with morphine and a dose-response inhibition relationship can be seen. All the doses of morphine suppressed the formalininduced paw lick behaviour of the rats (Fig. 2). For comparative purposes the reduction in behavioural score has been calculated and averaged over a 20 min period immediately following the second peak in the behavioural score. The percentage reductions were 23.6%, 23.3% and 24.5% for 1.5, 3 and 5 mg/kg morphine respectively. The test is sensitive to all the doses of morphine, yet fails to distinguish between them.
Electrical stimulation of the APtN The effects of electrical stimulation of the APtN in the 4 analgesiometric tests are illustrated in Fig. 3. This shows that APtN stimulation reduces responses to noxious stimuli in the tail-flick, paw pressure and formalin tests but had no detectable effect in the hot-plate test. The tail-flick test was most sensitive to the effects of APtN stimulation which was applied at time 0 with 35 PA r.m.s. 50 Hz sine wave current for 15 sec. The tail-flick latency was maximal immediately following stimulation and then slowly decreased but was not at baseline within the test period. A similar experimental protocol was used for the paw pressure tests. APtN stimulation caused an immediate increase in the pressure required to evoke a response which was maintained for 35 min. The effect was never maximal, however, and was shorter lasting than the effects of APtN stimulation in the tail-flick test. The hot-plate test (+ in Fig. 3A) showed no antinociceptive effect of APtN stimulation. In contrast to the tail-flick and paw pressure tests, a consistent reduction in the response latency was seen although at no time was this a significant deviation from baseline levels. The effects of intradermal formalin on behaviour were very much reduced by APtN stimulation for a period of 10 min (Fig. 3B). Subsequently the behaviours
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Fig. 2. The effectiveness of systemic morphine in causing antinociception measured by the formalin test. Each group contained 8 rats. Saline-treated animals (A) illustrate the effects of intradermal formalin. Formalin was administered before time - 15. The behavioural score was immediately high, subsided for a few minutes and then peaked again at about 0 min to remain above normal levels for about 50 min. Just prior to time 0 subcutaneous injections of morphine were made. 5 (A), 3 (a) and 1.5 (0) mg/kg morphine all attenuated the behavioural score, all by approximately the same amount. * Significant difference from control values (P < 0.05).
of the stimulated
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It is clear that APtN stimulation, applied for 15 sec. had effects which were less long lasting than the effects of any dose of morphine, in all the tests. in the tail-flick test both morphine and APtN stimulation caused equivalent and maximal inhibition of responses. Similarly. in the formalin test APtN stimulation and all doses of morphine caused a potent and equivalent inhibition of the behavioural responses. In the paw pressure test APtN stimulation was less effective than 5 mg/kg morphine but had the same effect as the lower doses. The extremely variable and rather weak effects of morphine in the hot-plate test were not mimicked at all by APtN stimulation. Histology Following experiments involving APtN stimulation the animals were killed and the brains removed and stored in formal-saline. Stimulation sites were identified from 50 pm brain sections cut on a freezing microtome. The data from stimulation sites outside the APtN were excluded from this study.
Discussion
were identical.
Comparison of the effects of morphine and APtN stimuiation The analgesiometric tests differ profoundly from each other in the extent to which they are affected by morphine. This may indicate some biological selectivity in the actions of morphine but is also 1ikeIy to be due to differences in the sensitivity of the tests to analgesic treatments. It is necessary, therefore, to compare the effects of APtN stimulation on each test with the effect of morphine on each test.
These data have confirmed the previously reported antinociceptive effects of APtN stimulation on the tailflick reflex to noxious heat. This test is frequently criticised because it is a spinal reflex which is displayed by normal and spinal animals and those anaesthetised by barbiturates. It may well be a poor test for analgesic treatments which act at supraspinal levels. A controversial aspect of the tail-flick test concerns the brevity of the stimulus. Many of the clinically important pains are not sharp or brief in character but are chronic and of a
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Fig. 3. A: effectiveness of APtN stimulation in causing antinociception in the tail-flick (A), paw pressure (A) and hot-plate (+) tests. At time 0 a 15 set period of 35 pA sine wave current was applied to the APtN. Following stimulation, the tail-flick latency immediately increased to maximal. It remained high for the following 20 mm and then gradually declined to baseline by 50 min. APtN stimulation also increased the vocabsation latency in the paw pressure test, the increase is half maximal and peaks at 15-20 mm before gradually declining to baseline. In the hot-plate test, however, no antinc&eptive effect is seen. B: effects of APtN stimtdation in the formalin test. After stimulation at time 0 there is a large decrease in the behavioural score which persists for 5-10 min. * Significant difference from control values (P i 0.05).
199
burning character. These are the characteristics of the second pain following formalin injection reported by volunteers. It is clearly important to determine the effectiveness of APtN stimulation against a range of noxious stimuli of varying types. To make such comparisons meaningful, however, it has to be recognised that behavioural tests vary not only in the nature of the noxious stimulus or the type of response, but also in the variability of the response and therefore in the overall sensitivity of the test. In the present study this problem has been approached by comparing the effectiveness of APtN stimulation on each test to the potency of morphine. APtN stimulation and morphine were both very effective in the tail-flick and formalin test. Both treatments caused similar increases in vocalisation latency in the paw pressure test but in the very variable hot-plate test, morphine displayed a moderate potency and APtN stimulation was without effect. This lack of effect in the hot-plate test is not due to APtN stimulation being ineffective against thermally noxious stimuli, for APtN stimulation potently inhibits responses in the tail-flick response to noxious heat. Neither can it be concluded that APtN stimulation is less effective than morphine when a centrally coordinated response is the end-point of the test, for both the paw pressure and formalin tests involve coordinated responses. It is likely that some other aspect of the detailed application of the hot-plate test explains its relative lack of sensitivity to both the effects of morphine and APtN stimulation. Morphine has been shown to inhibit preferentially the second phase of the response to subdermal formalin and it has been suggested that this second phase is due to activation of C fibres [1,5]. The present data show that APtN stimulation potently inhibits behavioural responses to clinically important C fibre activation. The data shown in Fig. 3B do not seem to imply a very large inhibition of behavioural score but comparison with Fig. 2 shows that a similar degree of inhibition was seen with morphine. The apparent magnitude of the effect of both treatments will of course reflect the arbitrarily chosen method of scoring the behaviour. That all doses of morphine were initially equally effective suggests that the test is very sensitive to morphine. It may be concluded from these studies that on a range of nociceptive tests APtN stimulation has effects which are broadly parallel to those of morphine within the dose range 3-5 mg/kg. This confirms the strong and long lasting antinociceptive effect of this stimulation. Previously, studies of APtN have examined only inhibition of the tail-flick response to noxious heat [12,15]. Using this technique it has been shown that the effects of APtN stimulation may be blocked by naloxone, alpha ,-adrenoceptor antagonists and atropine but not 5-HT antagonists [ll]. The lack of effect of 5-HT antagonists suggests that
the serotonergic raphe-spinal axons do not mediate the inhibitory effects of APtN stimulation, and this has been confirmed by the observation that NRM lesions do not block the effects of APtN stimulation [14]. Other studies have shown that the APtN comprises a central relay of the important ascending effects of dorsal column stimulation [13]. It is quite likely that the valuable clinical effects of dorsal column stimulation are mediated via the APtN, and the present data confirm that the antinociception evoked from APtN is effective against a range of noxious stimuli which may have greater clinical relevance than that applied in the tailflick test. The inhibition of responses to subcutaneously injected formalin is also of interest because Porro et al. [lo] have shown that subcutaneously administered formalin significantly elevates the metabolic rate (and presumably electrical activity) of cells in the APtN. Thus it may be suggested that the activation of peripheral nociceptors by formalin injection activates cells in the APtN which, possibly by inhibiting dorsal horn neurones, reduces the defensive response of the animal to formalin.
References 1 Banna, N.R., Saade, N.E., Atweh, S.F. and Jabbur, S.J., Prolonged discharge of wide dynamic range spinal neurons evoked by formaldehyde injected in their cutaneous receptive fields, Exp. Neurol., 93 (1986) 275-278. J.O. and Sessle, B.J.. Inhibi2 Chiang, C.Y.. Chen, I.C., Dostrovsky. tory effects of anterior pretectal nucleus stimulation on jaw opening reflex and trigeminal (V) brainstem neuronal responses in rats, Sot. Neurosci. Abst., 13 (1989) 272.1. 3 Cohen, S.R. and Melzack, R.. Habenular stimulation produces analgesia in the formalin test, Neurosci. Lett.. 70 (1986) 165-169. 4 Dennis, S.G. and Melzack. R., Pain signalling systems in the dorsal and ventral cord, Pain, 4 (1977) 97-132. A.H. and Sullivan, A.F., Subcutaneous formalin-in5 Dickenson, duced activity of dorsal horn neurones in the rat: differential response to an intrathecal opiate administered pre or post formalin, Pain, 30 (1987) 349-360. 6 Foster, G.A., Rees, H., Roberts, M.H.T. and Sizer, A.R., Microinjection of tetracaine into the anterior pretectal nucleus blocks the antinociceptive effects of deep mesencephalic stimulation in the anaesthetised rat, J. Physiol. (Lond.), 412 (1989) 21P. I Irwin. S., Honde. R.W., Bennett, D.R.. Hendershot, L.C. and Seevers, M.H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. Pharmacol. Exp. Ther.. 10 (1951) 132-143. 8 Ogren, S.O. and Holm, A.C., Test-specific effects of the 5-HT reuptake inhibitors alaproclate and zimelidine on pain sensitivity and morphine analgesia, J. Neural Transm., 47 (1980) 253-271. 9 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 10 Porro, C.A., Cavazzuti, M.. Biral, G.P. and Galetti, A., Acute pain increases metabolic activity in the rat anterior pretectal nucleus, Pain, Suppl. 4 (1987) S403. 11 Rees, H.. Prado. W.A., Rawlings, S. and Roberts, M.H.T., The effects of intraperitoneal administration of antagonists and development of morphine tolerance on the antinociception produced by
12
13
14
15
stimulating the anterior pretectal nucleus of &he rat, Br. J. Pharmacol., 92 (1987) 769-779. Rees, H. and Roberts, M.H.T.. Anterior pretectal stimulation alters the responses of spinal dorsal horn neurones to cutaneous stimulation in the rat, J. Physiol. (Lond.), 385 (1987) 415-436. Rees, H. and Roberts. M.H.T.. Supraspinally mediated antinociceptive effects of dorsal column stimulation in the rat: involvement of the anterior pretectal nucleus, J. Physiol. (Lond.), 417 (1989) 375-388. Rees, H., Wilson, D.G. and Roberts, M.H.T.. Analgesia evoked from the posterior diencephalon (anterior pretectal nucleus), Pain, Suppl. 4 (1987) s33. Roberts, M.H.T. and Rees, H., The antinociceptive effects of stimulating the pretectal nucleus of the rat, Pain, 25 (1986) 83-93.
16 Tyers. M.B., A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol., 69 (1980) 503-512. 17 Vierck, C.J. and Cooper, B.Y., Guidelines for assessing pain reactions and pain modulation in laboratory animals. In: L. Kruger and J.C. Liebeskind (Eds.), Advances in Pain Research and Therapy, Vol. 6. Raven Press, New York, 1984, pp. 305-322. 18 Yaksh, T.L., Multiple spinal opiate receptors in analgesia. In: 1~. Kruger and J.C. Liebeskind (Eds.), Advances in Pain Research and Therapy, Vol. 6, Raven Press, New York, 1984, pp. 197-215. 19 Yaksh, T.L., Rodham, N.R.F. and Jensen, T.S., Sites of action of opiates in production of analgesia. In: H.L. Fields and J.-M. Besson (Eds.), Progress in Brain Research, Vol. 77, Elsevier Science Publishers, Amsterdam, 1988, pp. 371-394.
Pain, 44 (1991) 195-200 0 1991 Elsevier Science Publishers B.V. 0304-3959/91/%03.50 ADONIS
030439599100077M
PAIN 01731
The antinociceptive effects of anterior pretectal stimulation in tests using thermal, mechanical and chemical noxious stimuli D.G. Wilson, H. Rees and M.H.T. Roberts Depamnem
of Physk&gy,
Uniuersity of Wales College of Cardi& Cardiff CFI ISS (U.K.)
(Received 19 March 1990, revision received 29 June 1990, accepted 14 August 1990)
Summary
Four behavioural tests have been used to study the antinociceptive effects of electrical stimulation of the anterior pretectal nucleus (APtN) in the rat. The antinociceptive effects of stimulating this nucleus, which lies dorsally in the posterior diencephalon, have recently been studied extensively but always using briefly applied heat stimuli. It is reported here that APtN stimulation effectively inhibited responses to briefly applied noxious pressure and longer-lasting noxious chemical (formalin) stimuli. Although the tail-flick reflex to noxious heat was very potently depressed by APtN stimulation, responses to noxious heat in the hot-plate test were not. Three doses of morphine were also studied with each test and it was concluded that 15 set of 35 PIA r.m.s. current into the APtN was as effective as 3-5 mg/kg morphine S.C.in the rat. Key words: Pain; Antinociception;
Paw pressure test; Formalin test; Hot plate; Tail flick
Stimulation of the anterior pretectal nucleus (APtN) has been shown to inhibit potently the tail-flick reflex to noxious heat without disrupting motor behaviour or responses to non-noxious stimuli fl5]. The stimulation is not aversive. The APtN is an import~t relay of the antinociceptive effects of stimulating the somatosensory cortex [2], the dorsal columns [13] and deep mesencephalic nuclei [6]. Spinal lamina V cell responses to noxious thermal stimuli are inhibited by APtN stimulation. The descending inhibitory pathway runs in the dorsolateral funiculus. All these data have been obtained using briefly applied thermal stimuli and the behavioural data have all been obtained with studies of the spinal reflex ‘tail-flick’ response. The tail flick does not require the integrative actions of higher centres [7] and its sole use as a means of identifying antinociception has been questioned [l&19]. We have therefore studied the effects of electrical stimulation of the APtN
Corres~omfenee to: Dr. H. Rees, Department of Physjology, University of Wales College of Cardiff, PO Box 902, Cardiff CFl lSS, U.K.
on behaviours which are thought to be mediated supraspinally. Furthermore, antinociceptive tests employing brief stimuli which give sensations of sharp pain to human volunteers may be affected differently by analgesics from those tests which give sensations of prolonged burning pain. The effects of APtN stimulation on both types of test have been studied. The antinociceptive tests which have been employed are: (1) the hot-plate test, (2) the paw pressure test, and (3) the formalin test. In addition, the sensitivities of these tests to APtN stimulation have been compared to the tail-flick test. For the purpose of comparison the sensitivities of the tests, as used in this laboratory, to varying doses of morphine have also been investigated.
Methods and experimental
design
One hundred male Wistar rats (270-290 g) were used. The animals were divided into two groups, one group was used in studies which involved electrical stimulation of the APtN and the other to compare the effects of morphine. Sixty-four rats were used in the study of the effects of morphine, they were divided into 4 groups of 16,
each group being used to study responses to a different analgesiometric test. The animals were tested on 2 separate occasions separated by at least 1 week. Animals received a single subcutaneous injection of either 1.5, 3, or 5 mg/ kg morphine or 0.05 ml saline. On the second test the animal received a different dose of morphine or saline. Procedures for the implantation of guide cannulae, the tail-flick test and for electrical stimulation of the APtN have been described in details elsewhere [15]. Briefly, 12 mm lengths of hypoder~c needle tubing were stereotaxically positioned into a hole drilled through the skull and held in place by dental acrylic. The end of the cannula lay 3 mm above the APtN. A unipolar insulated steel wire was cut to 15 mm and inserted into the cannula so that the tip lay in the APtN. Fifty Hz sine wave current of 35 PA r.m.s. was applied to the electrode for 15 sec. Four analgesiometric tests were used. The tail-flick procedure has been described elsewhere [15]. Briefly, the rat was placed into a clear glass tube from which the tail protruded. A part of the tail between 2 and 6 cm from the tip was placed onto a nichrome wire coil at room temperature. A calibrated current was passed through the coil so as to raise its temperature 9” C/set. After about 3 set (coil temperature about 45’C) normal rats flicked the tail away from the heat source. If no response occurred the tail was removed after 6 set to prevent tissue damage. Scores were normalised according to the following formula: Index of analgesia =
latency - control latency 6 - control latency
The control latency was the average of 3 responses immediately preceding the injection or APtN stimulation. The paw pressure test used the ‘Ugo Basile Analgesy-meter.’ Each rat was gently held with a hind paw placed under a pressure pad. The force applied to the paw was progressively increased, at a rate of 48 g/set to a maximum weight of 750 g or until either a vocalisation or struggle was evoked. The pressure was immediately removed and the force required to elicit the end-point response was noted. Data were normal&d in the same way as described for the tail-flick test, except that the pressure applied to achieve the end-point was used as the score. Baseline levels were also determined as before. In the formalin test the general methods outlined by Cohen and Melzack [3] have been used with a few minor alterations. The test involved the use of clear glass or perspex observation chambers with mirrors placed at an angle underneath them. The behaviour of the rats was examined and scored for 30 set periods at 5 min intervals both before and after the intradermal injection of 0.05 ml of a 2.5% formalin solution into the
ptantar surface of the right hind paw. The score was based on the position and utilisation of the right hind paw. Normal weight-bearing behaviour was assigned a score of ‘0.’ whilst the shaking or licking of the paw was assigned a ‘3.’ Scores of ‘1’ and ‘2’ represent a gradation between the two extremes. The scores were fed into an on-line computer, which was used in the analysis of the data. Intradermal formalin caused an increase in behavioural scores at 5 min followed by a rapid decline to just above normal score levels 5 min later. This was foBowed by a second rise in scores which was sustained for 20 min before gradually declining. This response was consistent for all control animals and is in agreement with other studies using this analgesiometric test [3,4]. The experimental variable was applied prior to the second increase in behavioural score. The first increase was too variable to allow meaningful comparisons, slight differences in time between formalin injection and recording periods affect the magnitude of the recorded peak giving variable rest&s. In contrast. the second peak in behavioural score provides a prolonged steady plateau response and this response has been taken by others to represent tonic pain [1,5]. In the hot-plate test the general methods outlined by others have been followed [11,16]. The hot-plate test comprised a high-sided enamel box held semi-submerged in a heated water bath. The water temperature was thermostatically controlled so that the enamel surface was at 55°C. The animal was placed on to the plate and the period of time which elapsed until a hind paw was licked was recorded. The paw lick was taken to be the end-point of the test, and the rat was immediately removed from the box. If the animal did not lick the hind paw within the 30 set test period it was removed from the hot plate to prevent tissue damage. Determinations of statistical significance were done using the non-parametric Mann-Whitney U test.
Results
Behavioural tests conducted in different laboratories or with different strains of rats vary greatly with respect to the effectiveness of analgesic treatments. Standard doses of morphine have been administered in order to define the sensitivity of these tests to this well known analgesic and to provide a baseline for comparison of the effects of APtN stimulation. Effects of subcutaneous morphine Subcutaneously administered morphine at 5, 3 and 1.5 mg/kg caused a rapid elevation of tail-flick latency to maximal or near maximal levels. The effects were shorter lasting for the lower doses but, as illustrated by Fig. lA, complete recovery of baseline latencies did not occur for any dose within 90 min of the injection.
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Fig. 1. A: the effectiveness of 3 doses of morphine in causing inhibition of the tail-flick reflex to noxious heat. Calculation of the index of analgesia is described in the text. Subcutaneous injections of morphine 5 mg/kg (A), 3 mg/kg (+) or 1.5 mg/kg (0) or saline (A) were made just prior to time 0. Each group contained 8 animals. All doses of morphine were effective but the lower doses acted for a shorter period. Saline was without effect. B: the antinociceptive effect of subcutaneous morphine in the paw pressure test. Each group contained 8 animals. All doses of morphine increased the vocalisation latency. The increase was gradual in onset, maximal effects being observed 20-50 min post injection. The greatest increase was seen following an administration of 5 mg/kg morphine (A). There was little difference between the (+) and 1.5 (0) mg/kg doses. No increase was seen with saline-treated animals. C: the antinociceptive potency of morphine in the hot-plate test. Each group contained 8 animals. 5 (A), 3 (+) and 1.5 (0) mg/kg morphine all increased the paw lick latency. Mine (A) had no effect. The large standard error bars indicate the considerable variability of the weak antinociceptive effect of morphine. * Significant difference from control values (P i 0.05).
The paw pressure test was less sensitive to the effects of morphine and the variability was much greater. The data are shown in Fig. 1B. Maximal scores were not achieved except for one point with the highest dose. The onset of the effects of morphine was much slower than with the tail-flick test and peak effects were seen 50 min after the injection. The duration of the effect of morphine seemed to be less and baseline levels were nearly recovered 90 min after the injection. The greatest effect was seen with the higher dose but the test did not discriminate between the two lower doses of morphine. The results obtained with the hot-plate test (illustrated in Fig. 1C) show even greater variability than the paw pressure test. The hot-plate test is not usually used to display the full time course of an analgesic effect and it is clear from the data why this is so. Nevertheless, a clear inhibition of the response latency occurred with morphine and a dose-response inhibition relationship can be seen. All the doses of morphine suppressed the formalininduced paw lick behaviour of the rats (Fig. 2). For comparative purposes the reduction in behavioural score has been calculated and averaged over a 20 min period immediately following the second peak in the behavioural score. The percentage reductions were 23.6%, 23.3% and 24.5% for 1.5, 3 and 5 mg/kg morphine respectively. The test is sensitive to all the doses of morphine, yet fails to distinguish between them.
Electrical stimulation of the APtN The effects of electrical stimulation of the APtN in the 4 analgesiometric tests are illustrated in Fig. 3. This shows that APtN stimulation reduces responses to noxious stimuli in the tail-flick, paw pressure and formalin tests but had no detectable effect in the hot-plate test. The tail-flick test was most sensitive to the effects of APtN stimulation which was applied at time 0 with 35 PA r.m.s. 50 Hz sine wave current for 15 sec. The tail-flick latency was maximal immediately following stimulation and then slowly decreased but was not at baseline within the test period. A similar experimental protocol was used for the paw pressure tests. APtN stimulation caused an immediate increase in the pressure required to evoke a response which was maintained for 35 min. The effect was never maximal, however, and was shorter lasting than the effects of APtN stimulation in the tail-flick test. The hot-plate test (+ in Fig. 3A) showed no antinociceptive effect of APtN stimulation. In contrast to the tail-flick and paw pressure tests, a consistent reduction in the response latency was seen although at no time was this a significant deviation from baseline levels. The effects of intradermal formalin on behaviour were very much reduced by APtN stimulation for a period of 10 min (Fig. 3B). Subsequently the behaviours
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Fig. 2. The effectiveness of systemic morphine in causing antinociception measured by the formalin test. Each group contained 8 rats. Saline-treated animals (A) illustrate the effects of intradermal formalin. Formalin was administered before time - 15. The behavioural score was immediately high, subsided for a few minutes and then peaked again at about 0 min to remain above normal levels for about 50 min. Just prior to time 0 subcutaneous injections of morphine were made. 5 (A), 3 (a) and 1.5 (0) mg/kg morphine all attenuated the behavioural score, all by approximately the same amount. * Significant difference from control values (P < 0.05).
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It is clear that APtN stimulation, applied for 15 sec. had effects which were less long lasting than the effects of any dose of morphine, in all the tests. in the tail-flick test both morphine and APtN stimulation caused equivalent and maximal inhibition of responses. Similarly. in the formalin test APtN stimulation and all doses of morphine caused a potent and equivalent inhibition of the behavioural responses. In the paw pressure test APtN stimulation was less effective than 5 mg/kg morphine but had the same effect as the lower doses. The extremely variable and rather weak effects of morphine in the hot-plate test were not mimicked at all by APtN stimulation. Histology Following experiments involving APtN stimulation the animals were killed and the brains removed and stored in formal-saline. Stimulation sites were identified from 50 pm brain sections cut on a freezing microtome. The data from stimulation sites outside the APtN were excluded from this study.
Discussion
were identical.
Comparison of the effects of morphine and APtN stimuiation The analgesiometric tests differ profoundly from each other in the extent to which they are affected by morphine. This may indicate some biological selectivity in the actions of morphine but is also 1ikeIy to be due to differences in the sensitivity of the tests to analgesic treatments. It is necessary, therefore, to compare the effects of APtN stimulation on each test with the effect of morphine on each test.
These data have confirmed the previously reported antinociceptive effects of APtN stimulation on the tailflick reflex to noxious heat. This test is frequently criticised because it is a spinal reflex which is displayed by normal and spinal animals and those anaesthetised by barbiturates. It may well be a poor test for analgesic treatments which act at supraspinal levels. A controversial aspect of the tail-flick test concerns the brevity of the stimulus. Many of the clinically important pains are not sharp or brief in character but are chronic and of a
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Fig. 3. A: effectiveness of APtN stimulation in causing antinociception in the tail-flick (A), paw pressure (A) and hot-plate (+) tests. At time 0 a 15 set period of 35 pA sine wave current was applied to the APtN. Following stimulation, the tail-flick latency immediately increased to maximal. It remained high for the following 20 mm and then gradually declined to baseline by 50 min. APtN stimulation also increased the vocabsation latency in the paw pressure test, the increase is half maximal and peaks at 15-20 mm before gradually declining to baseline. In the hot-plate test, however, no antinc&eptive effect is seen. B: effects of APtN stimtdation in the formalin test. After stimulation at time 0 there is a large decrease in the behavioural score which persists for 5-10 min. * Significant difference from control values (P i 0.05).
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burning character. These are the characteristics of the second pain following formalin injection reported by volunteers. It is clearly important to determine the effectiveness of APtN stimulation against a range of noxious stimuli of varying types. To make such comparisons meaningful, however, it has to be recognised that behavioural tests vary not only in the nature of the noxious stimulus or the type of response, but also in the variability of the response and therefore in the overall sensitivity of the test. In the present study this problem has been approached by comparing the effectiveness of APtN stimulation on each test to the potency of morphine. APtN stimulation and morphine were both very effective in the tail-flick and formalin test. Both treatments caused similar increases in vocalisation latency in the paw pressure test but in the very variable hot-plate test, morphine displayed a moderate potency and APtN stimulation was without effect. This lack of effect in the hot-plate test is not due to APtN stimulation being ineffective against thermally noxious stimuli, for APtN stimulation potently inhibits responses in the tail-flick response to noxious heat. Neither can it be concluded that APtN stimulation is less effective than morphine when a centrally coordinated response is the end-point of the test, for both the paw pressure and formalin tests involve coordinated responses. It is likely that some other aspect of the detailed application of the hot-plate test explains its relative lack of sensitivity to both the effects of morphine and APtN stimulation. Morphine has been shown to inhibit preferentially the second phase of the response to subdermal formalin and it has been suggested that this second phase is due to activation of C fibres [1,5]. The present data show that APtN stimulation potently inhibits behavioural responses to clinically important C fibre activation. The data shown in Fig. 3B do not seem to imply a very large inhibition of behavioural score but comparison with Fig. 2 shows that a similar degree of inhibition was seen with morphine. The apparent magnitude of the effect of both treatments will of course reflect the arbitrarily chosen method of scoring the behaviour. That all doses of morphine were initially equally effective suggests that the test is very sensitive to morphine. It may be concluded from these studies that on a range of nociceptive tests APtN stimulation has effects which are broadly parallel to those of morphine within the dose range 3-5 mg/kg. This confirms the strong and long lasting antinociceptive effect of this stimulation. Previously, studies of APtN have examined only inhibition of the tail-flick response to noxious heat [12,15]. Using this technique it has been shown that the effects of APtN stimulation may be blocked by naloxone, alpha ,-adrenoceptor antagonists and atropine but not 5-HT antagonists [ll]. The lack of effect of 5-HT antagonists suggests that
the serotonergic raphe-spinal axons do not mediate the inhibitory effects of APtN stimulation, and this has been confirmed by the observation that NRM lesions do not block the effects of APtN stimulation [14]. Other studies have shown that the APtN comprises a central relay of the important ascending effects of dorsal column stimulation [13]. It is quite likely that the valuable clinical effects of dorsal column stimulation are mediated via the APtN, and the present data confirm that the antinociception evoked from APtN is effective against a range of noxious stimuli which may have greater clinical relevance than that applied in the tailflick test. The inhibition of responses to subcutaneously injected formalin is also of interest because Porro et al. [lo] have shown that subcutaneously administered formalin significantly elevates the metabolic rate (and presumably electrical activity) of cells in the APtN. Thus it may be suggested that the activation of peripheral nociceptors by formalin injection activates cells in the APtN which, possibly by inhibiting dorsal horn neurones, reduces the defensive response of the animal to formalin.
References 1 Banna, N.R., Saade, N.E., Atweh, S.F. and Jabbur, S.J., Prolonged discharge of wide dynamic range spinal neurons evoked by formaldehyde injected in their cutaneous receptive fields, Exp. Neurol., 93 (1986) 275-278. J.O. and Sessle, B.J.. Inhibi2 Chiang, C.Y.. Chen, I.C., Dostrovsky. tory effects of anterior pretectal nucleus stimulation on jaw opening reflex and trigeminal (V) brainstem neuronal responses in rats, Sot. Neurosci. Abst., 13 (1989) 272.1. 3 Cohen, S.R. and Melzack, R.. Habenular stimulation produces analgesia in the formalin test, Neurosci. Lett.. 70 (1986) 165-169. 4 Dennis, S.G. and Melzack. R., Pain signalling systems in the dorsal and ventral cord, Pain, 4 (1977) 97-132. A.H. and Sullivan, A.F., Subcutaneous formalin-in5 Dickenson, duced activity of dorsal horn neurones in the rat: differential response to an intrathecal opiate administered pre or post formalin, Pain, 30 (1987) 349-360. 6 Foster, G.A., Rees, H., Roberts, M.H.T. and Sizer, A.R., Microinjection of tetracaine into the anterior pretectal nucleus blocks the antinociceptive effects of deep mesencephalic stimulation in the anaesthetised rat, J. Physiol. (Lond.), 412 (1989) 21P. I Irwin. S., Honde. R.W., Bennett, D.R.. Hendershot, L.C. and Seevers, M.H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. Pharmacol. Exp. Ther.. 10 (1951) 132-143. 8 Ogren, S.O. and Holm, A.C., Test-specific effects of the 5-HT reuptake inhibitors alaproclate and zimelidine on pain sensitivity and morphine analgesia, J. Neural Transm., 47 (1980) 253-271. 9 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 10 Porro, C.A., Cavazzuti, M.. Biral, G.P. and Galetti, A., Acute pain increases metabolic activity in the rat anterior pretectal nucleus, Pain, Suppl. 4 (1987) S403. 11 Rees, H.. Prado. W.A., Rawlings, S. and Roberts, M.H.T., The effects of intraperitoneal administration of antagonists and development of morphine tolerance on the antinociception produced by
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stimulating the anterior pretectal nucleus of &he rat, Br. J. Pharmacol., 92 (1987) 769-779. Rees, H. and Roberts, M.H.T.. Anterior pretectal stimulation alters the responses of spinal dorsal horn neurones to cutaneous stimulation in the rat, J. Physiol. (Lond.), 385 (1987) 415-436. Rees, H. and Roberts. M.H.T.. Supraspinally mediated antinociceptive effects of dorsal column stimulation in the rat: involvement of the anterior pretectal nucleus, J. Physiol. (Lond.), 417 (1989) 375-388. Rees, H., Wilson, D.G. and Roberts, M.H.T.. Analgesia evoked from the posterior diencephalon (anterior pretectal nucleus), Pain, Suppl. 4 (1987) s33. Roberts, M.H.T. and Rees, H., The antinociceptive effects of stimulating the pretectal nucleus of the rat, Pain, 25 (1986) 83-93.
16 Tyers. M.B., A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol., 69 (1980) 503-512. 17 Vierck, C.J. and Cooper, B.Y., Guidelines for assessing pain reactions and pain modulation in laboratory animals. In: L. Kruger and J.C. Liebeskind (Eds.), Advances in Pain Research and Therapy, Vol. 6. Raven Press, New York, 1984, pp. 305-322. 18 Yaksh, T.L., Multiple spinal opiate receptors in analgesia. In: 1~. Kruger and J.C. Liebeskind (Eds.), Advances in Pain Research and Therapy, Vol. 6, Raven Press, New York, 1984, pp. 197-215. 19 Yaksh, T.L., Rodham, N.R.F. and Jensen, T.S., Sites of action of opiates in production of analgesia. In: H.L. Fields and J.-M. Besson (Eds.), Progress in Brain Research, Vol. 77, Elsevier Science Publishers, Amsterdam, 1988, pp. 371-394.