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Unilateral analgesia produced by intraventricular morphine
Brain Research, 303 (1984) 277-287 Elsevier
277
BRE 10105
Unilateral Analgesia Produced by IntraventricularMorphine S. ROBIN COHEN, FRANCES V. ABBOTI" and RONALD MELZACK
Department of Psychology, McGill University, Montreal, Quebec (Canada) (Accepted November 15th, 1983)
Key words: morphine - - laterality - - lateral ventricle - - analgesia - - formalin test - - foot-flick test - - pain
Morphine injected into the lateral ventricle of the rat produced unilateral analgesia in the formalin test, which involves continuous, moderate pain. In contrast, analgesia was produced bilaterally in the foot-flick test which involves brief, rapidly rising pain. In the formalin test, intraventricular morphine produced analgesia in the ipsilateral but not the contralateral hindpaw. Analgesia was achieved with relatively low doses of morphine (2.5-10.0 k~g)in the formalin test while very high doses (50-200/~g) were necessary to produce analgesia in the foot-flick test. These results add to other data indicating that different neural mechanisms underlie opiate analgesia in different types of pain. Moreover, they indicate that, in the formalin test, the neural mechanisms of morphine analgesia are somatotopically organized and that forebrain structures are likely to be involved.
INTRODUCTION Recent studies suggest that different neural structures are involved in the analgesic effects of morphine on different kinds of pain. Generally, the neural structures responsible for analgesia in tests which evoke brief, rapidly rising pain, such as the tail-flick and pinch tests, seem to lie in the brainstem. In these tests, opiates appear to produce analgesia by activating brainstem structures which project to the dorsal horns of the spinal cord and inhibit incoming nociceprive information. The pathway which has been most extensively investigated projects from the periaqueductal gray matter (PAG) in the midbrain to the nucleus raphe magnus (NRM), which in turn projects through the dorsolateral funiculus (DLF) to the dorsal horns of the spinal cord (for reviews see refs. 20 and 30). Other brainstem structures implicated in morphine analgesia in these pain tests are the nucleus reticularis paragigantocellularis4,7, 36 and the nucleus reticularis gigantocellularis4,16,26. In contrast, different structures appear to be predominantly involved in producing analgesia in a test of continuous, moderate pain such as the formalin
test. In this test, pain is produced by injecting dilute formalin under the skin of a rat's paw. The pain response consists of favoring the injected paw, lifting it offthe ground, and at times licking or chewing it. The pain appears to be moderate and lasts about 2 h. There is now increasing evidence that, when the pain of the formalin test is studied, ascending projections to the forebrain are more important than descending projections to the spinal cord. Destruction of the N R M or the caudal P A G attenuates analgesia produced by stimulation in the midbrain or by morphine in the tail-flick test, but has no effect on stimulationproduced or morphine analgesia in the formalin test2,3. Furthermore, while a D L F lesion attenuates morphine analgesia in the tail-flick test, it has no effect on morphine analgesia in the formalin test (L. Watkins, personal communication, 1983). In addition, destruction of the median raphe nucleus (MR) has no effect in the tail-flick test1,15,28,44, while in the formalin test, rats with a lesion in the M R show a potentiation of morphine analgesia or, possibly, a reduction in the amount of pain produced by the formalint,3. Similarly, the threshold to elicit a prolonged squeal by stimulation of the trigeminal nerve is great-
Correspondence: S. R. Cohen, Department of Psychology, McGill University, 1205 Docteur Penfield Avenue, Montreal, Quebec, Canada H3A 1B1. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
278 ly increased in rats after a lesion in the MR and dorsal raphe 44. Unlike the NRM, which projects to the spinal cord 9, the MR projects largely to forebrain structuresl3.17. Using the pinch test, there have been several reports of patterns of cutaneous analgesia which reveal a somatotopic organization in the underlying mechanisms. Yaksh et al. al report that injections of morphine into the rostral PAG of rats produced analgesia in the face and forepaws but not in the trunk or hindpaws. When pinches were applied within 10 min after microinjection into some of these sites, analgesia was restricted to the ipsilateral face and forepaws. After 10 min the analgesia was always bilateral. Somatotopic organization is also revealed by electrical stimulation of discrete points in the PAG. Usually analgesia is elicited over the whole body or bilaterally in restricted areas of the body31.38,39,42. Less often, analgesia is limited to only one side of the body, either ipsilateral or contralateral to the stimulating electrode. While Soper 3s and Mayer and Liebeskind 29 report stimulation-produced analgesia restricted to the contralateral side of the body, Basbaum et a1.11 report stimulation-produced analgesia only on the ipsilateral side. Although studies have been carried out on the effects of intracerebroventricular (i.c.v.) microinjection of opiates on brief, rapidly rising pain, no one has studied the effects of i. c.v. opiates on continuous, moderate pain, such as that produced in the formalin test. Injection of morphine into a lateral ventricle produces analgesia in the tail-flick and hot plate tests14,25,35,40, but no tests were performed to determine whether the pattern of analgesia shows any type of somatotopic organization. Furthermore, this procedure has never been carried out with the formalin test. Consequently, a pilot study was conducted in which morphine was injected into the lateral ventricle of a rat, and its effects on formalin-induced pain were recorded. Surprisingly, morphine produced analgesia when it was injected into the lateral ventricle ipsilateral to the formalin-injected hindpaw but had no effect when it was injected into the contralateral ventricle. This study investigates the laterality of analgesia after i.c.v, injection of morphine using the formalin test and the foot-flick test, which was used in place of the tail-flick test in order to permit testing of laterality effects.
MATERIALSAND METHODS
Subjects and housing The subjects were male hooded rats of the LongEvans strain from the Charles River barrier-sustained colony. They were housed 3 or 4 to a cage in the colony room on a 14/10 h light/dark cycle. Food and water were available ad libitum throughout the experiment.
Surgery The rats weighed 250-330 g at the time of surgery. An outer cannula constructed from a 23-gauge needle was implanted under sodium pentobarbital anaesthesia above the lateral ventricle (1.2 mm posterior and 2.8 mm lateral to bregma, and 3.2 mm below the skull surface with the incisor bar set 5.0 mm above the interaural line33). A stylet which extended just beyond the tip of the outer cannula was inserted into it and left in place until the time of testing, at least 1 week later.
Drugs and intraventricular injection procedure For i.c.v, injection, morphine sulphate was dissolved in sterile normal saline. All doses were calculated on the basis of the salt. In control conditions, saline alone was injected. After the rat was removed from the formalin test box, or after baseline testing in the foot-flick test, the stylet was removed and an inner cannula was inserted into the outer cannula. The inner cannula was a 30gauge stainless steel tube which extended 1.5 mm beyond the outer cannula. The inner cannula was connected to a Sage Instruments infusion pump by PE10 tubing. Either saline or morphine was injected in a volume of 5 or 15/A over 1 min. The inner cannula was left in place for 1 min after the injection and then removed, the stylet replaced, and the rat returned to the formalin test box or, in the foot-flick test, to the home cage. If fluid appeared at the top of the outer cannula at any time during the injection or during testing, the data from that rat were discarded.
Testing procedure and apparatus Formalin test. Each rat was placed in the test box at least 20 min prior to the formalin injection to allow habituation to the apparatus. The box was made of
279 clear Plexiglas, and was 32 x 32 × 32 cm in size. Below the floor, a mirror angled at 45 ° allowed the experimenter an unobstructed view of the rat's paws. After habituation, the plantar surface of the hindpaw either ipsilateral or contralateral to the cannulated ventricle was injected subcutaneously with 0.05 ml of 2.5% formalin. The paw to be injected was determined randomly. The formalin injection produces an apparently moderate pain which diminishes in 5-10 min and then rises to a steady level about 20 rain
after injection and lasts about 1.5 h. Therefore, tests of the effects of i.c.v, injection took place during the period when pain is stable. Thirty rain after the formalin injection, the rat was removed from the test box and was injected intraventricularly with either saline or morphine (2.5 #g, 5.0/~g, or 10.0/~g) in a volume of 5 #1. The rat was replaced in the box and pain rating began 40 rain after formalin injection and was continued for 50 rain. The behavioral pain rating scale used by Dubuisson and Dennism was modified
Fig. 1. The behavioral categories and the corresponding pain rating values in the formalin test when the hindpaw is injected. The pain rating values and behaviors are: 0, walking or sitting normally; 1, walking or sitting favoring the injected paw (note that the rat's paw is not flat on the ground); 2, lifting the injected paw off the ground; 3, licking or chewing the injected paw.
280 for use with the hindpaw and the categories are illustrated in Fig. I. A weighted pain score for each 10 rain block was obtained by multiplying the n u m b e r of seconds spent in each category by the pain rating value, adding these scores from all categories, and dividing the total by 600 s to obtain a pain score with a possible range of 0.0 to 3.0. Catalepsy test. A t the end of 50 rain of pain rating, the rat was r e m o v e d from the formalin test box and tested for catalepsy. The rat's hindpaws were placed on the table and the forepaws on a b a r 10 cm above the table. The time for the rat to remove both forepaws from the bar was measured. This p r o c e d u r e was repeated 3 times. If a rat r e m a i n e d with forepaws on the bar for 60 s he was given a score of 60 s for that trial, removed from the bar, and replaced on it for the next trial. The catalepsy score for each rat was the mean of the 3 trials. Foot-flick test. The rat was hand-held, with nose towards the wrist of the e x p e r i m e n t e r and hindpaws on the experimenter's fingertips. The hindpaw ipsilateral or contralateral to the cannula was quickly dipped into a b e a k e r of water maintained at 50 ° + 0.5 °C. The amount of time for the rat to flick his paw out of the hot water was recorded. This was r e p e a t e d for the other hindpaw. A cut-off time of 12 s was used to avoid burning the paw. H a l f of the rats at each drug dose had the hindpaw ipsilateral to the cannula tested first and the other half had the contralateral hindpaw tested first. A f t e r recording a baseline foot-flick latency, the rats were injected intraventricularly with either saline or morphine (25fig, 5 0 y g , 100yg, or 200fig) in a volume of 5 ,ul. These higher doses were used because pilot studies indicated that lower doses, such as those used in the formalin test, were ineffective in the foot-flick test at this injection site. A n additional group of rats received 50/~g of m o r p h i n e in a volume of 15/A, while another group received 15 ~1 of saline. The rats were then tested for foot-flick latency 15, 30, 45, 60 and 90 rain after i.c.v, injection. Between trials they were returned to their h o m e cage.
TABLE I Number of rats per group in the formalin test
Drug dose and side of body relative to icy injection are indicated. Different rats were used in each group. Drug
Formalin injected paw relative to cannula
Number of rats
Saline
ipsilateral contralateral ipsilateral contralateral ipsilateral contralateral ipsilateral contralateral
8 7 8 7 9 7 6 7
2.5#g morphine 5.0/~g morphine lO.O/*gmorphine
i.c.v, injection of m o r p h i n e at each dose level and the paw which was injected with formalin are shown in Table I. In the foot-flick test, b o t h hindpaws of each rat were tested. The n u m b e r of rats in each group that received different doses of morphine, and the volume of injection, are shown in Table II. The results from the formalin test and the footflick test with a 5 fll injection were separately analysed with 3-way A N O V A s . The square roots of the formalin pain scores and foot-flick latencies were used since the variances were correlated with the means. The i n d e p e n d e n t variables were time after i.c.v, injection, drug dose, and side of the body relative to the cannula. Because each rat contributed 5 scores in the formalin test (five 10-min time blocks) and the foot-flick test (5 test periods after i.c.v, injection), these sets of scores were treated as a r e p e a t e d measure in each A N O V A . F u r t h e r m o r e , in the
TABLE II Number of rats per group in the foot-flick test
Drug dose and volume of injection are indicated. Different rats were used in each group. Both hindpaws of each rat were tested. Volume of injection
Drug
Number of rats
5/A
Saline 25/,tg morphine 50/ag morphine 100/~g morphine 200 ~g morphine
8 4 9 9 10
15 fll
Saline 50/~g morphine
3 5
Design and data analys&
Separate groups of rats were used in the formalin and foot-flick tests. Each rat was tested with only one drug dose. In the formalin test, only one hindpaw of each rat was tested. The n u m b e r of rats that received
281 A N O V A for the foot-flick test, the side of the body relative to the cannula was treated as a repeated measure since data for the ipsilateral and contralateral hindpaws were obtained from the same group of rats. The effect of a larger volume of injection in the foot-flick test was evaluated by comparing the square roots of the foot-flick latencies for the group which received 50 gg of morphine in 5 #1 of saline with those of the group that received the same dose of morphine in 15 /~1 of saline. A 3-way, 2-repeated measure A N O V A was performed with time of testing, volume of injection, and side relative to cannula as the independent variables. The logarithms of the catalepsy scores were analyzed with a 2-way A N O V A for independent groups for the variables Drug and Side.
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The rats were deeply anaesthetized with chloral hydrate. The inner cannula was inserted and 5 #1 of India ink were injected by the infusion pump over a period of 1 min. During the injection the rat's head was gently shaken to simulate normal movement. The inner cannula was removed 1 min later and the stylet was replaced. After 4-10 min, the rat was perfused intracardially with 0.9% saline followed by 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. The brain was sliced in several coronal sections, and visual inspection of the location of the ink and the cannula tip showed clearly whether or not the microinjected fluid successfully reached the ventricular system. Only when the ink was visible throughout the third and fourth ventricles and the injected lateral ventricle was that rat included in the study. In successful cases, the cannulae were usually located between the following coordinates33: AP --1.0 to --1.5; L---2.5 to --3.5; DV --2.7 to ---4.0 mm. RESULTS The results show clearly (Fig. 2) that, in the formalin test, injection of morphine into the lateral ventricle ipsilateral to the injured hindpaw produced analgesia. In contrast, morphine injected into the contralateral lateral ventricle had little or no analgesic effect. Generally, pain scores decreased during the 50
T
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10-20 20-30 30-40 40-50 50-50
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10-20 20-30 30-40 40-50 50-60
TIME AFTER INTRAVENTRICULAR INJECTION (rain)
Fig. 2. Time course of pain scores in the formalin test. Note that in order to make this figure comparablewith those for footflick test, less pain (or more analgesia) is represented towards the top of the graph. Mean pain score + S.E.M. for the hindpaws ipsilateral (O) and contralateral (~7) to the i.c.v, injection are illustrated for each of the 10 rain time blocks comprising the 50 min test period. The separate graphs show the effect of i.c.v, injection of saline, 2.5/lg, 5.0 #g, and 10.0/~g of morphine, respectively.
min testing period in rats which received either saline or morphine intraventricularly [F(4,204) = 48.13, P < 0.001]. However, morphine produced a significantly greater decrease in pain [F(3,51) = 3.06, P < 0.05]. Furthermore, the effect of the morphine was much stronger when the hindpaw ipsilateral to the cannula was injured than when the hindpaw contralateral to the cannula was injured [F(1,51) = 4.22, P < 0.05]. There were no 2- or 3-way interactions. The results obtained with the foot-flick test (Fig. 3) are strikingly different from the formalin test results. While morphine produced analgesia [F(4,35) = 7.14, P < 0.001] and this analgesia increased over time [F(4,140) = 4.57, P < 0.005], there was no difference in the withdrawal latencies between the hindpaws ipsilateral and contralateral to the cannula [F(1,35) = 1.45, P > 0.20] at any dose level of drug. There were no 2- or 3-way interactions. It is evident from Fig. 3 that a high dose of mor-
282
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TIME AFTER INTRAVENTRICULAR INJECTION (MIN) Fig. 3. Time course of paw withdrawal latencies in the foot-flick test. Mean paw withdrawal latencies + S.E.M. for the hindpaws ipsilateral (@) and contralateral (~7) to the i.c.v, injection at each time tested are illustrated. The separate graphs show the effect of i.c.v, injection of saline, 25/~g, 50/~g, 100/~g, and 200/~g of morphine, respectively.
phine was required to produce analgesia in the footflick test, since 25/,g of morphine had no analgesic effect. Because high doses of morphine were necessary, it is possible that the morphine was injected far from its site of action, so that only a small proportion of the injected morphine was reaching the active site. To determine whether this explanation is plausible, 50/~g of morphine were injected in a volume of 15/zl rather than 5/,1, since injection in a larger volume allows more morphine to diffuse away from the site of injection. Therefore, the analgesic effect of 50 /,g of morphine injected in 5/,1 and in 15/,1 of saline were compared. Fig. 4 shows that morphine administered in a volume of 15 /~1 produced significantly more analgesia than when administered in a volume of 5 pl [F(1,12) = 7.48, P < 0.05]. At all times of testing, the rats which received the morphine in 15/~1 of saline were more analgesic than those which received the same amount of morphine in 5/*1 of saline. There
was no difference in the amount of analgesia shown by the hindpaws ipsilateral and contralateral to the cannula [F(1,12) = 1.27, P > 0.20]. As before, the analgesia increased over time [F(4,48) = 2.83, P < 0.05]. There were no 2- or 3-way interactions. Fig. 4 also shows that 15/*1 of saline alone did not affect the baseline foot-flick latencies. It is clear from Fig. 3 that, in the foot:flick test, i.c.v. morphine produces its maximal effect about 45 rain after the injection. The changing baseline in the formalin test makes it difficult to determine the peak effect. Fig. 5 shows dose-effect curves for both pain tests using the 45-min foot-flick (peak effect) and the pain scores for the comparable period (40-50 min) in the formalin test. In Fig. 5, both the pain scores and foot-flick latencies are expressed as a percentage of the combined saline-injected groups' scores for the relevant time period. While the regression lines were generated from the responses of individual rats, for
283 o 5Jul saline v 151JI saline • 501Jg morphine in 5ul ,saline • 501Jg morphine in 15111 saline
l/t
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10.0 25.0 50.0 MORPHINE DOSE (pg)
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Fig. 5. Dose-effect relationships in the formalin and foot-flick tests. The results are expressed as percentage of combined (ipsilateral + contralateral) saline mean. Means are taken from the period of peak effect in the foot-flick test (45 rain after i.c.v. injection) and the comparable period in the formalin test (40-50 min after i.c.v, injection). Separate regression lines for the ipsilateral and contralateral paws were fitted for each pain test.
o
DISCUSSION
o--
I
Baseline TIME AFTER
i
15
I
30
I
45
INTRAVENTRICULAR
I
60
I
90
INJECTION (rain)
Fig. 4. Effect of increased volume of injection in the foot-flick test. The effects of 50 #g of morphine in 5/~1 (O) and 15 #1 ( T ) of saline on foot-flick latency are compared. Group mean + S.E.M. is plotted for each time of testing. The data from injection of vehicle alone are also illustrated. The effect of the i.c.v, injection on the ipsilateral hindpaw is shown at the top, that on the contralateral hindpaw is at the bottom.
the purpose of clarity only the mean responses are shown. This treatment of the data emphasizes the marked lateralized analgesia in the formalin test and the difference in sensitivity of the two tests to morphine administered into the lateral ventricle. Fig. 6 shows the catalepsy scores of rats immediately after the formalin test. While morphine produced a d o s e - d e p e n d e n t c a t a l e p s y [ F ( 3 , 5 0 ) = 7.20, P <
0.001],
Morphine injected into the lateral ventricle produced analgesia in both the formalin and foot-flick tests. In the formalin test, the analgesia was significantly greater when the i.c.v, injection was ipsilateral to the formalin-injected paw than when it was contralateral. There was no evidence of lateralized effects in the foot-flick test. The formalin test was also much more sensitive than the foot-flick test to morphine administered by this route. The discovery of morphine's unilateral analgesic effect in the formalin test was unexpected. It is reasonable to assume that injection of morphine into the
"~ 4 0 . 0 - - u)
~
IPSILATERAL PAW INJECTED CONTRALATERAL PAW INJECTED
U,I nO ¢n 10.0>u~ n LU .-J ,< I-,< 0 2.0.
t h e d e g r e e of c a t a l e p s y w a s t h e s a m e
whether the injured hindpaw was ipsilateral or cont r a l a t e r a l t o t h e c a n n u l a [ F ( 1 , 5 0 ) = 0.93, P > 0.30]. There was no significant Drug x Side interaction. T h e c a t a l e p s y s c o r e f o r o n e r a t in t h e i p s i l a t e r a l saline g r o u p was n o t a v a i l a b l e .
Saline
2.5
5
10
MORPHINE D O S E (Pg)
Fig. 6. Catalepsy in the formalin test groups. Catalepsy was measured 60 min after i.c.v, injection. Mean catalepsy score + S.E.M. is shown for each Drug × Side group.
284 lateral ventricle would produce a lateralized effect only if the morphine acted at a site near the lateral ventricle rather than near the third and fourth ventricles. If the analgesia were due to diffusion of morphine into the third or fourth ventricle, a unilateral effect would not be expected. It can be seen in Fig. 5 that in the formalin test the 5 ~g and 10 ktg doses of morphine had similar effects on the hindpaw ipsilateral to the i.c.v, injection. This possibly reflects a ceiling in the degree of analgesia which can be obtained in the formalin test from morphine injected intraventricularly rather than directly into brain tissue. Alternatively, while a dose higher than 10 ~g may produce a greater degree of analgesia, pilot studies indicated that at higher doses the difference between the ipsilateral and contralateral hindpaws diminished, since analgesia appeared in the contralateral hindpaw. The analgesia in the contralateral hindpaw after larger doses is probably due to diffusion of morphine to the opposite hemisphere. Because data from the paws ipsilateral and contralateral to the cannula were collected from separate groups of rats in the formalin test, it is important that these groups were equally cataleptic, as indicated by the bar test. Since the demonstration of pain in the formalin test is dependent on the ability to perform actions such as lifting or licking a hindpaw, a difference in catalepsy between groups might be interpreted as a difference in analgesia. However, in this case this does not present a problem since groups with the ipsilateral hindpaw injected displayed a degree of catalepsy comparable to that in groups with the contralateral hindpaw injected. While others have studied the effects of microinjection of opiates in the forebrain, they did not report a resultant analgesia. Microinjection of morphine into the caudate nucleus, anterior thalamus, medial thalamus, and septum did not produce analgesia 41,43. However, the pain tests used in these studies involved brief, rapidly rising pain, and, as this experiment and others >3 have shown, the brain structures involved in analgesia in these tests are probably different from those in the formalin test. The forebrain structures responsible for the analgesia produced in the formalin test are as yet unknown. It is possible that morphine may produce analgesia by acting on the striatum, and especially on the caudate nucleus. The caudate forms a large part
of the lateral wall of the lateral ventricle. Furthermore, several studies report that in the rat brain parts of the caudate are rich in opiate receptors 6,32,34. Electrical stimulation of the caudate has been reported to raise the pain threshold in monkeys37 and to alleviate chronic pain in man 22. Other structures possibly involved in the analgesic action of morphine in this experiment are the interstitial nucleus of the stria terminalis, nucleus accumbens, and the medial part of the lateral habenula. Each of these structures is located, at least in part, near the lateral ventricle and all contain a fairly large number of opiate receptors 6,32,34. In addition, the interstitial nucleus of the stria terminalis and lateral habenula receive projections from the MR but the nucleus accumbens and caudate do not13. This may be important since rats with a lesion in the MR show a potentiation of morphine analgesia or a reduction in the amount of pain produced in the formalin test1, 3. The participation of forebrain structures in morphine analgesia in formalin test pain does not mean that brainstem structures play no role in this analgesia. Possibly the forebrain structures produce their effects through action on brainstem structures, which in turn, as discussed in the introduction with respect to brief, rapidly rising pain, may act to inhibit incoming pain signals at the spinal level. However, in these pain tests supraspinal structures are thought to send efferents through the DLF to inhibit pain signals at the spinal level8d2, 20.23. Yet destruction of the DLF has no effect on morphine analgesia in the formalin test (L. Watkins, personal communication, 1983). Therefore, if brainstem structures are important for morphine analgesia in the formalin test, they play a role different from that in brief, rapidly rising pain. Alternatively, the analgesia produced by morphine's action on forebrain structures may not depend on a descending system at all. The morphine may act at forebrain levels to block or alter incoming nociceptive signals so that they are no longer perceived as painful. Amodei and PaxinosS, using unilateral knife cuts through the medial forebrain bundle and the medial internal capsule and/or the thalamus, have already provided evidence that nociceptive signals produced by a formalin injection are processed in the ipsilateral forebrain. It is possible that i.c.v. morphine relieves pain in the formalin test in a manner similar to that in which frontal lobotomy or cingu-
285 lotomy relieve chronic pain in humans. Both frontal lobotomy and cingulotomy appear to leave the sensory aspect of pain intact, but greatly reduce the anxiety and suffering that accompany chronic pain 18,21. In the foot-flick test, morphine produced bilateral analgesia. This does not mean that the neural mechanism through which morphine produces its analgesic effect in the foot-flick test has no somatotopic organization with respect to laterality. The results simply indicate that, with this method of morphine administration, no lateralization of effect is present. It is noteworthy that Yaksh et al. 41 report analgesia briefly restricted to the ipsilateral face and forepaw after injection of morphine into the rostral PAG using the pinch test. Very high doses of morphine were required to produce an increase in foot-flick latency. Pilot studies conducted with low doses, similar to those used in the formalin test, indicated that they did not affect the foot-flick latency. It can be seen in Fig. 3 that even 25 #g of morphine had no effect on foot-flick latency. Yet the foot-flick test is sensitive to the analgesic effect of systemic morphine. Studies in this laboratory have shown that 12 mg/kg of morphine produce a sizeable increase in latency to withdraw the paw from hot water. Using focused light as a source of heat, Levine et al. 27 report that the foot-flick is similar to the tail-flick and responds similarly to systemic morphine. If the structures responsible for morphine analgesia in tests measuring brief, rapidly rising pain are in the brainstem, near the third and fourth ventricles and the aqueduct of Sylvius, as has been suggested by others a°,2°,3°, then it is possible that very high doses of morphine are needed in the foot-flick test to enable a sufficient amount of morphine to reach these brainstem structures. This suggestion is supported by the fact that analgesia was increased by simply increasing the volume of the microinjection, which would be expected to increase the diffusion of the morphine from the injection site. In fact, Herz et al. 24, measuring the latency of a licking reaction produced by stimulating the tooth-pulp in the rabbit, report that i.c.v, morphine restricted to the lateral and third ventricles produces no analgesia, but morphine restricted to the aqueduct and fourth ventricle produces analgesia. In addition, Van Ree 40, using the hot plate test in rats, reports that when the i.c.v, injection is directed at the lateral or third ventricles, 4 times more morphine is required to produce analge-
sia than when the morphine is injected into the fourth ventricle. There is another indication that morphine did not act, in the foot-flick test, on structures close to the lateral ventricle. With doses of 50/2g and 100 pg of morphine, the analgesia did not reach a maximum until 30-45 min after i.c.v, injection, suggesting that the morphine had to diffuse from the lateral ventricle before it could produce its maximal effect. With the 200 pg dose, the maximal effect was attained 15 min after injection, at the first time of testing. This may reflect the arbitrary ceiling on paw withdrawal latency, since at this dose and time of measurement there were several rats that left their paws in the water for the maximal 12 s. Herz et al. 24 using tooth-pulp stimulation in the rabbit, report that the maximal analgesic effect is reached more quickly after injection of morphine into the aqueduct than into the lateral ventricle. In conclusion, the data reveal that the system through which i.c.v, morphine exerts its analgesic effect on continuous, moderate pain is organized so that each hemisphere modulates pain in the ipsilateral but not the contralateral hindpaw. Although it is clear that a considerable degree of lateralization exists, it is possible that each hemisphere also exerts minor control over continuous, moderate pain in the contralateral hindpaw. Possibly, this lateralization applies to the entire body, but further experimentation is necessary to demonstrate this. No evidence was found for a similar somatotopic organization for modulation of brief, rapidly rising pain. Furthermore, the data strongly suggest that forebrain structures near the lateral ventricle participate in morphine analgesia for continuous, moderate pain. The data also suggest that the neural structures responsible for morphine analgesia in brief, rapidly rising pain are different from those responsible for morphine's effect on continuous, moderate pain. ACKNOWLEDGEMENTS The results of this study were published in abstract form in Society for Neuroscience Abstracts, 1983. This research was supported by Natural Sciences and Engineering Research Council of Canada Grant A7891 to Dr. Ronald Melzack. S. R. Cohen is supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship.
286 REFERENCES 1 Abbott, F. V. and Melzack, R., Brainstem lesions dissociate neural mechanisms of morphine analgesia in different kinds of pain, Brain Research, 251 (1982) 149-155. 2 Abbott, F. V. and Melzack, R., Dissociation of the mechanisms of stimulation-produced analgesia in tests of tonic and phasic pain. In J. J. Bonica, U. Lindblom and A. Iggo (Eds.), Advances in Pain Research and Therapy, Vol. 5, Raven Press, New York, 1983, pp. 401-409. 3 Abbott, F. V., Melzack, R. and Samuel, C., Morphine analgesia in the tail-flick and formalin pain tests is mediated by different neural systems, Exp. Neurol., 75 (1982) 644--651. 4 Akaike, A., Shibata, T., Satoh, M. and Takagi, H., Analgesia induced by microinjection of morphine into, and electrical stimulation of, the nucleus reticularis paragigantocellularis of rat medulla oblongata, Neuropharmacology, 17 (1978) 775-778. 5 Amodei, N. and Paxinos, G., Unilateral knife cuts produce ipsilateral suppression of responsiveness to pain in the formalin test, Brain Research, 193 (1980) 85-94. 6 Atweh, S. F. and Kuhar, M. J., Autoradiographic localization of opiate receptors in rat brain. III. The telencephalon, Brain Research, 134 (1977) 393-405. 7 Azami, J., Llewelyn, M. B. and Roberts, M. H. T., The contribution of nucleus reticularis paragigantocellularis and nucleus raphe magnus to the analgesia produced by systemically administered morphine, investigated with the microinjection technique, Pain, 12 (1982) 229-246. 8 Barton, C., Basbaum, A. I. and Fields, H. L., Dissociation of supraspinal and spinal actions of morphine: a quantitative evaluation, Brain Research, 188 (1980) 487-498. 9 Basbaum, A. I., Clanton, C. H. and Fields, H. L., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems, J. comp. Neurol., 178 (1978) 209-224. 10 Basbaum, A. I. and Fields, H. L., Endogenous pain control mechanisms: review and hypothesis, Ann. Neurol., 4 (1978) 451-462. 11 Basbaum, A. I., Marley, N. and O'Keefe, J., Spinal cord pathways involved in the production of analgesia by brain stimulation. In J. J. Bonica and D. Albe-Fessard (Eds.), Advances in Pain Research and Therapy, Vol. 1, Raven Press, New York, 1976, pp. 511-515. 12 Basbaum, A. I., Marley, N. J. E., O'Keefe, J. and Clanton, C. H., Reversal of morphine and stimulus-produced analgesia by subtotal spinal cord lesions, Pain, 3 (1977) 43-56. 13 Bobillier, P., Seguin, S., Petitjean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Research, 113 (1976) 449-486. 14 Brady, L. S. and Holtzman, S. G., Analgesic effects of intraventricular morphine and enkephalins in nondependent and morphine-dependent rats, J. Pharmacol. exp. Ther., 222 (1982) 190-197. 15 Buxbaum, D. M., Yarbrough, G. G. and Carter, M. E., Biogenic amines and narcotic effects. I. Modification of morphine-induced analgesia and motor activity after alteration of cerebral amine levels, J. Pharm'acol. exp. Ther., 185 (1973) 317-327. 16 Chan, S. H. H., Participation of the nucleus reticularis gigantocellularis in the morphine suppression of jaw-opening reflex in cats, Brain Research, 160 (1979) 377-380.
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287
36
37
38
39
40
its infusion into different liquor spaces in rat brain, Arch. Int. Pharmacodyn. Ther., 246 (1980) 108-117. Rosenfeld, J. P. and Stocco, S., Effects of midbrain, bulbar and combined morphine microinjections and systemic injections on orofacial nociception and rostral trigeminal stimulation: independent midbrain and bulbar opiate analgesia systems?, Brain Research, 215 (1981) 342-348. Schmidek, H. H., Fohanno, D., Ervin, F. R. and Sweet, W. H., Pain threshold alterations by brain stimulation in the monkey, J. Neurosurg., 35 (1971) 715-722. Soper, W. Y., Effects of analgesic midbrain stimulation on reflex withdrawal and thermal escape in the rat, J. cornp. physiol. Psychol., 90 (1976) 91-101. Soper, W. Y. and Melzack, R., Stimulation-produced analgesia: evidence for somatotopic organization in the midbrain, Brain Research, 251 (1982) 301-311. Van Ree, J. M., Multiple brain sites involved in morphine antinociception, J. Pharm. Pharmacol., 29 (1977) 765-767.
41 Yaksh, T. L., Yeung, J. C. and Rudy, T. A., Systematic examination in the rat of brain sites sensitive to the direct application of morphine: observation of differential effects within the periaqueductal gray, Brain Research, 114 (1976) 83-103. 42 Yeung, J. C., Yaksh, T. L. and Rudy, T. A., Concurrent mapping of brain sites for sensitivity to the direct application of morphine and focal electrical stimulation in the production of antinociception in the rat, Pain, 4 (1977) 23--40. 43 Yeung, J. C., Yaksh, T. L. and Rudy, T. A., Effect on the nociceptive threshold and EEG activity in the rat of morphine injected into the medial thalamus and the periaqueductal gray, Neuropharmacology, 17 (1978) 525-532. 44 York, J. L. and Maynert, E. W., Alterations in morphine analgesia produced by chronic deficits of brain catecholamines or serotonin: role of analgesimetric procedure, Psychopharmacology, 56 (1978) 119--125.
277
BRE 10105
Unilateral Analgesia Produced by IntraventricularMorphine S. ROBIN COHEN, FRANCES V. ABBOTI" and RONALD MELZACK
Department of Psychology, McGill University, Montreal, Quebec (Canada) (Accepted November 15th, 1983)
Key words: morphine - - laterality - - lateral ventricle - - analgesia - - formalin test - - foot-flick test - - pain
Morphine injected into the lateral ventricle of the rat produced unilateral analgesia in the formalin test, which involves continuous, moderate pain. In contrast, analgesia was produced bilaterally in the foot-flick test which involves brief, rapidly rising pain. In the formalin test, intraventricular morphine produced analgesia in the ipsilateral but not the contralateral hindpaw. Analgesia was achieved with relatively low doses of morphine (2.5-10.0 k~g)in the formalin test while very high doses (50-200/~g) were necessary to produce analgesia in the foot-flick test. These results add to other data indicating that different neural mechanisms underlie opiate analgesia in different types of pain. Moreover, they indicate that, in the formalin test, the neural mechanisms of morphine analgesia are somatotopically organized and that forebrain structures are likely to be involved.
INTRODUCTION Recent studies suggest that different neural structures are involved in the analgesic effects of morphine on different kinds of pain. Generally, the neural structures responsible for analgesia in tests which evoke brief, rapidly rising pain, such as the tail-flick and pinch tests, seem to lie in the brainstem. In these tests, opiates appear to produce analgesia by activating brainstem structures which project to the dorsal horns of the spinal cord and inhibit incoming nociceprive information. The pathway which has been most extensively investigated projects from the periaqueductal gray matter (PAG) in the midbrain to the nucleus raphe magnus (NRM), which in turn projects through the dorsolateral funiculus (DLF) to the dorsal horns of the spinal cord (for reviews see refs. 20 and 30). Other brainstem structures implicated in morphine analgesia in these pain tests are the nucleus reticularis paragigantocellularis4,7, 36 and the nucleus reticularis gigantocellularis4,16,26. In contrast, different structures appear to be predominantly involved in producing analgesia in a test of continuous, moderate pain such as the formalin
test. In this test, pain is produced by injecting dilute formalin under the skin of a rat's paw. The pain response consists of favoring the injected paw, lifting it offthe ground, and at times licking or chewing it. The pain appears to be moderate and lasts about 2 h. There is now increasing evidence that, when the pain of the formalin test is studied, ascending projections to the forebrain are more important than descending projections to the spinal cord. Destruction of the N R M or the caudal P A G attenuates analgesia produced by stimulation in the midbrain or by morphine in the tail-flick test, but has no effect on stimulationproduced or morphine analgesia in the formalin test2,3. Furthermore, while a D L F lesion attenuates morphine analgesia in the tail-flick test, it has no effect on morphine analgesia in the formalin test (L. Watkins, personal communication, 1983). In addition, destruction of the median raphe nucleus (MR) has no effect in the tail-flick test1,15,28,44, while in the formalin test, rats with a lesion in the M R show a potentiation of morphine analgesia or, possibly, a reduction in the amount of pain produced by the formalint,3. Similarly, the threshold to elicit a prolonged squeal by stimulation of the trigeminal nerve is great-
Correspondence: S. R. Cohen, Department of Psychology, McGill University, 1205 Docteur Penfield Avenue, Montreal, Quebec, Canada H3A 1B1. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
278 ly increased in rats after a lesion in the MR and dorsal raphe 44. Unlike the NRM, which projects to the spinal cord 9, the MR projects largely to forebrain structuresl3.17. Using the pinch test, there have been several reports of patterns of cutaneous analgesia which reveal a somatotopic organization in the underlying mechanisms. Yaksh et al. al report that injections of morphine into the rostral PAG of rats produced analgesia in the face and forepaws but not in the trunk or hindpaws. When pinches were applied within 10 min after microinjection into some of these sites, analgesia was restricted to the ipsilateral face and forepaws. After 10 min the analgesia was always bilateral. Somatotopic organization is also revealed by electrical stimulation of discrete points in the PAG. Usually analgesia is elicited over the whole body or bilaterally in restricted areas of the body31.38,39,42. Less often, analgesia is limited to only one side of the body, either ipsilateral or contralateral to the stimulating electrode. While Soper 3s and Mayer and Liebeskind 29 report stimulation-produced analgesia restricted to the contralateral side of the body, Basbaum et a1.11 report stimulation-produced analgesia only on the ipsilateral side. Although studies have been carried out on the effects of intracerebroventricular (i.c.v.) microinjection of opiates on brief, rapidly rising pain, no one has studied the effects of i. c.v. opiates on continuous, moderate pain, such as that produced in the formalin test. Injection of morphine into a lateral ventricle produces analgesia in the tail-flick and hot plate tests14,25,35,40, but no tests were performed to determine whether the pattern of analgesia shows any type of somatotopic organization. Furthermore, this procedure has never been carried out with the formalin test. Consequently, a pilot study was conducted in which morphine was injected into the lateral ventricle of a rat, and its effects on formalin-induced pain were recorded. Surprisingly, morphine produced analgesia when it was injected into the lateral ventricle ipsilateral to the formalin-injected hindpaw but had no effect when it was injected into the contralateral ventricle. This study investigates the laterality of analgesia after i.c.v, injection of morphine using the formalin test and the foot-flick test, which was used in place of the tail-flick test in order to permit testing of laterality effects.
MATERIALSAND METHODS
Subjects and housing The subjects were male hooded rats of the LongEvans strain from the Charles River barrier-sustained colony. They were housed 3 or 4 to a cage in the colony room on a 14/10 h light/dark cycle. Food and water were available ad libitum throughout the experiment.
Surgery The rats weighed 250-330 g at the time of surgery. An outer cannula constructed from a 23-gauge needle was implanted under sodium pentobarbital anaesthesia above the lateral ventricle (1.2 mm posterior and 2.8 mm lateral to bregma, and 3.2 mm below the skull surface with the incisor bar set 5.0 mm above the interaural line33). A stylet which extended just beyond the tip of the outer cannula was inserted into it and left in place until the time of testing, at least 1 week later.
Drugs and intraventricular injection procedure For i.c.v, injection, morphine sulphate was dissolved in sterile normal saline. All doses were calculated on the basis of the salt. In control conditions, saline alone was injected. After the rat was removed from the formalin test box, or after baseline testing in the foot-flick test, the stylet was removed and an inner cannula was inserted into the outer cannula. The inner cannula was a 30gauge stainless steel tube which extended 1.5 mm beyond the outer cannula. The inner cannula was connected to a Sage Instruments infusion pump by PE10 tubing. Either saline or morphine was injected in a volume of 5 or 15/A over 1 min. The inner cannula was left in place for 1 min after the injection and then removed, the stylet replaced, and the rat returned to the formalin test box or, in the foot-flick test, to the home cage. If fluid appeared at the top of the outer cannula at any time during the injection or during testing, the data from that rat were discarded.
Testing procedure and apparatus Formalin test. Each rat was placed in the test box at least 20 min prior to the formalin injection to allow habituation to the apparatus. The box was made of
279 clear Plexiglas, and was 32 x 32 × 32 cm in size. Below the floor, a mirror angled at 45 ° allowed the experimenter an unobstructed view of the rat's paws. After habituation, the plantar surface of the hindpaw either ipsilateral or contralateral to the cannulated ventricle was injected subcutaneously with 0.05 ml of 2.5% formalin. The paw to be injected was determined randomly. The formalin injection produces an apparently moderate pain which diminishes in 5-10 min and then rises to a steady level about 20 rain
after injection and lasts about 1.5 h. Therefore, tests of the effects of i.c.v, injection took place during the period when pain is stable. Thirty rain after the formalin injection, the rat was removed from the test box and was injected intraventricularly with either saline or morphine (2.5 #g, 5.0/~g, or 10.0/~g) in a volume of 5 #1. The rat was replaced in the box and pain rating began 40 rain after formalin injection and was continued for 50 rain. The behavioral pain rating scale used by Dubuisson and Dennism was modified
Fig. 1. The behavioral categories and the corresponding pain rating values in the formalin test when the hindpaw is injected. The pain rating values and behaviors are: 0, walking or sitting normally; 1, walking or sitting favoring the injected paw (note that the rat's paw is not flat on the ground); 2, lifting the injected paw off the ground; 3, licking or chewing the injected paw.
280 for use with the hindpaw and the categories are illustrated in Fig. I. A weighted pain score for each 10 rain block was obtained by multiplying the n u m b e r of seconds spent in each category by the pain rating value, adding these scores from all categories, and dividing the total by 600 s to obtain a pain score with a possible range of 0.0 to 3.0. Catalepsy test. A t the end of 50 rain of pain rating, the rat was r e m o v e d from the formalin test box and tested for catalepsy. The rat's hindpaws were placed on the table and the forepaws on a b a r 10 cm above the table. The time for the rat to remove both forepaws from the bar was measured. This p r o c e d u r e was repeated 3 times. If a rat r e m a i n e d with forepaws on the bar for 60 s he was given a score of 60 s for that trial, removed from the bar, and replaced on it for the next trial. The catalepsy score for each rat was the mean of the 3 trials. Foot-flick test. The rat was hand-held, with nose towards the wrist of the e x p e r i m e n t e r and hindpaws on the experimenter's fingertips. The hindpaw ipsilateral or contralateral to the cannula was quickly dipped into a b e a k e r of water maintained at 50 ° + 0.5 °C. The amount of time for the rat to flick his paw out of the hot water was recorded. This was r e p e a t e d for the other hindpaw. A cut-off time of 12 s was used to avoid burning the paw. H a l f of the rats at each drug dose had the hindpaw ipsilateral to the cannula tested first and the other half had the contralateral hindpaw tested first. A f t e r recording a baseline foot-flick latency, the rats were injected intraventricularly with either saline or morphine (25fig, 5 0 y g , 100yg, or 200fig) in a volume of 5 ,ul. These higher doses were used because pilot studies indicated that lower doses, such as those used in the formalin test, were ineffective in the foot-flick test at this injection site. A n additional group of rats received 50/~g of m o r p h i n e in a volume of 15/A, while another group received 15 ~1 of saline. The rats were then tested for foot-flick latency 15, 30, 45, 60 and 90 rain after i.c.v, injection. Between trials they were returned to their h o m e cage.
TABLE I Number of rats per group in the formalin test
Drug dose and side of body relative to icy injection are indicated. Different rats were used in each group. Drug
Formalin injected paw relative to cannula
Number of rats
Saline
ipsilateral contralateral ipsilateral contralateral ipsilateral contralateral ipsilateral contralateral
8 7 8 7 9 7 6 7
2.5#g morphine 5.0/~g morphine lO.O/*gmorphine
i.c.v, injection of m o r p h i n e at each dose level and the paw which was injected with formalin are shown in Table I. In the foot-flick test, b o t h hindpaws of each rat were tested. The n u m b e r of rats in each group that received different doses of morphine, and the volume of injection, are shown in Table II. The results from the formalin test and the footflick test with a 5 fll injection were separately analysed with 3-way A N O V A s . The square roots of the formalin pain scores and foot-flick latencies were used since the variances were correlated with the means. The i n d e p e n d e n t variables were time after i.c.v, injection, drug dose, and side of the body relative to the cannula. Because each rat contributed 5 scores in the formalin test (five 10-min time blocks) and the foot-flick test (5 test periods after i.c.v, injection), these sets of scores were treated as a r e p e a t e d measure in each A N O V A . F u r t h e r m o r e , in the
TABLE II Number of rats per group in the foot-flick test
Drug dose and volume of injection are indicated. Different rats were used in each group. Both hindpaws of each rat were tested. Volume of injection
Drug
Number of rats
5/A
Saline 25/,tg morphine 50/ag morphine 100/~g morphine 200 ~g morphine
8 4 9 9 10
15 fll
Saline 50/~g morphine
3 5
Design and data analys&
Separate groups of rats were used in the formalin and foot-flick tests. Each rat was tested with only one drug dose. In the formalin test, only one hindpaw of each rat was tested. The n u m b e r of rats that received
281 A N O V A for the foot-flick test, the side of the body relative to the cannula was treated as a repeated measure since data for the ipsilateral and contralateral hindpaws were obtained from the same group of rats. The effect of a larger volume of injection in the foot-flick test was evaluated by comparing the square roots of the foot-flick latencies for the group which received 50 gg of morphine in 5 #1 of saline with those of the group that received the same dose of morphine in 15 /~1 of saline. A 3-way, 2-repeated measure A N O V A was performed with time of testing, volume of injection, and side relative to cannula as the independent variables. The logarithms of the catalepsy scores were analyzed with a 2-way A N O V A for independent groups for the variables Drug and Side.
~ONTRALATERALPAW INJECTED 0,,~
Saline
0.2--
0.6 0.8 1.0 LU nO
o
O~ Z < O_
z ...I < nO LL
1.2 1.4 "1.6
010JJg morphine
0.2-
The rats were deeply anaesthetized with chloral hydrate. The inner cannula was inserted and 5 #1 of India ink were injected by the infusion pump over a period of 1 min. During the injection the rat's head was gently shaken to simulate normal movement. The inner cannula was removed 1 min later and the stylet was replaced. After 4-10 min, the rat was perfused intracardially with 0.9% saline followed by 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. The brain was sliced in several coronal sections, and visual inspection of the location of the ink and the cannula tip showed clearly whether or not the microinjected fluid successfully reached the ventricular system. Only when the ink was visible throughout the third and fourth ventricles and the injected lateral ventricle was that rat included in the study. In successful cases, the cannulae were usually located between the following coordinates33: AP --1.0 to --1.5; L---2.5 to --3.5; DV --2.7 to ---4.0 mm. RESULTS The results show clearly (Fig. 2) that, in the formalin test, injection of morphine into the lateral ventricle ipsilateral to the injured hindpaw produced analgesia. In contrast, morphine injected into the contralateral lateral ventricle had little or no analgesic effect. Generally, pain scores decreased during the 50
T
I
I
f
0.40.60.81.0-
_1/I
1.21.41.6f
Perfusion and histology
2,5 ~Jg morphine
0.4-
10-20 20-30 30-40 40-50 50-50
,
,
,
10-20 20-30 30-40 40-50 50-60
TIME AFTER INTRAVENTRICULAR INJECTION (rain)
Fig. 2. Time course of pain scores in the formalin test. Note that in order to make this figure comparablewith those for footflick test, less pain (or more analgesia) is represented towards the top of the graph. Mean pain score + S.E.M. for the hindpaws ipsilateral (O) and contralateral (~7) to the i.c.v, injection are illustrated for each of the 10 rain time blocks comprising the 50 min test period. The separate graphs show the effect of i.c.v, injection of saline, 2.5/lg, 5.0 #g, and 10.0/~g of morphine, respectively.
min testing period in rats which received either saline or morphine intraventricularly [F(4,204) = 48.13, P < 0.001]. However, morphine produced a significantly greater decrease in pain [F(3,51) = 3.06, P < 0.05]. Furthermore, the effect of the morphine was much stronger when the hindpaw ipsilateral to the cannula was injured than when the hindpaw contralateral to the cannula was injured [F(1,51) = 4.22, P < 0.05]. There were no 2- or 3-way interactions. The results obtained with the foot-flick test (Fig. 3) are strikingly different from the formalin test results. While morphine produced analgesia [F(4,35) = 7.14, P < 0.001] and this analgesia increased over time [F(4,140) = 4.57, P < 0.005], there was no difference in the withdrawal latencies between the hindpaws ipsilateral and contralateral to the cannula [F(1,35) = 1.45, P > 0.20] at any dose level of drug. There were no 2- or 3-way interactions. It is evident from Fig. 3 that a high dose of mor-
282
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TIME AFTER INTRAVENTRICULAR INJECTION (MIN) Fig. 3. Time course of paw withdrawal latencies in the foot-flick test. Mean paw withdrawal latencies + S.E.M. for the hindpaws ipsilateral (@) and contralateral (~7) to the i.c.v, injection at each time tested are illustrated. The separate graphs show the effect of i.c.v, injection of saline, 25/~g, 50/~g, 100/~g, and 200/~g of morphine, respectively.
phine was required to produce analgesia in the footflick test, since 25/,g of morphine had no analgesic effect. Because high doses of morphine were necessary, it is possible that the morphine was injected far from its site of action, so that only a small proportion of the injected morphine was reaching the active site. To determine whether this explanation is plausible, 50/~g of morphine were injected in a volume of 15/zl rather than 5/,1, since injection in a larger volume allows more morphine to diffuse away from the site of injection. Therefore, the analgesic effect of 50 /,g of morphine injected in 5/,1 and in 15/,1 of saline were compared. Fig. 4 shows that morphine administered in a volume of 15 /~1 produced significantly more analgesia than when administered in a volume of 5 pl [F(1,12) = 7.48, P < 0.05]. At all times of testing, the rats which received the morphine in 15/~1 of saline were more analgesic than those which received the same amount of morphine in 5/*1 of saline. There
was no difference in the amount of analgesia shown by the hindpaws ipsilateral and contralateral to the cannula [F(1,12) = 1.27, P > 0.20]. As before, the analgesia increased over time [F(4,48) = 2.83, P < 0.05]. There were no 2- or 3-way interactions. Fig. 4 also shows that 15/*1 of saline alone did not affect the baseline foot-flick latencies. It is clear from Fig. 3 that, in the foot:flick test, i.c.v. morphine produces its maximal effect about 45 rain after the injection. The changing baseline in the formalin test makes it difficult to determine the peak effect. Fig. 5 shows dose-effect curves for both pain tests using the 45-min foot-flick (peak effect) and the pain scores for the comparable period (40-50 min) in the formalin test. In Fig. 5, both the pain scores and foot-flick latencies are expressed as a percentage of the combined saline-injected groups' scores for the relevant time period. While the regression lines were generated from the responses of individual rats, for
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Fig. 5. Dose-effect relationships in the formalin and foot-flick tests. The results are expressed as percentage of combined (ipsilateral + contralateral) saline mean. Means are taken from the period of peak effect in the foot-flick test (45 rain after i.c.v. injection) and the comparable period in the formalin test (40-50 min after i.c.v, injection). Separate regression lines for the ipsilateral and contralateral paws were fitted for each pain test.
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Fig. 4. Effect of increased volume of injection in the foot-flick test. The effects of 50 #g of morphine in 5/~1 (O) and 15 #1 ( T ) of saline on foot-flick latency are compared. Group mean + S.E.M. is plotted for each time of testing. The data from injection of vehicle alone are also illustrated. The effect of the i.c.v, injection on the ipsilateral hindpaw is shown at the top, that on the contralateral hindpaw is at the bottom.
the purpose of clarity only the mean responses are shown. This treatment of the data emphasizes the marked lateralized analgesia in the formalin test and the difference in sensitivity of the two tests to morphine administered into the lateral ventricle. Fig. 6 shows the catalepsy scores of rats immediately after the formalin test. While morphine produced a d o s e - d e p e n d e n t c a t a l e p s y [ F ( 3 , 5 0 ) = 7.20, P <
0.001],
Morphine injected into the lateral ventricle produced analgesia in both the formalin and foot-flick tests. In the formalin test, the analgesia was significantly greater when the i.c.v, injection was ipsilateral to the formalin-injected paw than when it was contralateral. There was no evidence of lateralized effects in the foot-flick test. The formalin test was also much more sensitive than the foot-flick test to morphine administered by this route. The discovery of morphine's unilateral analgesic effect in the formalin test was unexpected. It is reasonable to assume that injection of morphine into the
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whether the injured hindpaw was ipsilateral or cont r a l a t e r a l t o t h e c a n n u l a [ F ( 1 , 5 0 ) = 0.93, P > 0.30]. There was no significant Drug x Side interaction. T h e c a t a l e p s y s c o r e f o r o n e r a t in t h e i p s i l a t e r a l saline g r o u p was n o t a v a i l a b l e .
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284 lateral ventricle would produce a lateralized effect only if the morphine acted at a site near the lateral ventricle rather than near the third and fourth ventricles. If the analgesia were due to diffusion of morphine into the third or fourth ventricle, a unilateral effect would not be expected. It can be seen in Fig. 5 that in the formalin test the 5 ~g and 10 ktg doses of morphine had similar effects on the hindpaw ipsilateral to the i.c.v, injection. This possibly reflects a ceiling in the degree of analgesia which can be obtained in the formalin test from morphine injected intraventricularly rather than directly into brain tissue. Alternatively, while a dose higher than 10 ~g may produce a greater degree of analgesia, pilot studies indicated that at higher doses the difference between the ipsilateral and contralateral hindpaws diminished, since analgesia appeared in the contralateral hindpaw. The analgesia in the contralateral hindpaw after larger doses is probably due to diffusion of morphine to the opposite hemisphere. Because data from the paws ipsilateral and contralateral to the cannula were collected from separate groups of rats in the formalin test, it is important that these groups were equally cataleptic, as indicated by the bar test. Since the demonstration of pain in the formalin test is dependent on the ability to perform actions such as lifting or licking a hindpaw, a difference in catalepsy between groups might be interpreted as a difference in analgesia. However, in this case this does not present a problem since groups with the ipsilateral hindpaw injected displayed a degree of catalepsy comparable to that in groups with the contralateral hindpaw injected. While others have studied the effects of microinjection of opiates in the forebrain, they did not report a resultant analgesia. Microinjection of morphine into the caudate nucleus, anterior thalamus, medial thalamus, and septum did not produce analgesia 41,43. However, the pain tests used in these studies involved brief, rapidly rising pain, and, as this experiment and others >3 have shown, the brain structures involved in analgesia in these tests are probably different from those in the formalin test. The forebrain structures responsible for the analgesia produced in the formalin test are as yet unknown. It is possible that morphine may produce analgesia by acting on the striatum, and especially on the caudate nucleus. The caudate forms a large part
of the lateral wall of the lateral ventricle. Furthermore, several studies report that in the rat brain parts of the caudate are rich in opiate receptors 6,32,34. Electrical stimulation of the caudate has been reported to raise the pain threshold in monkeys37 and to alleviate chronic pain in man 22. Other structures possibly involved in the analgesic action of morphine in this experiment are the interstitial nucleus of the stria terminalis, nucleus accumbens, and the medial part of the lateral habenula. Each of these structures is located, at least in part, near the lateral ventricle and all contain a fairly large number of opiate receptors 6,32,34. In addition, the interstitial nucleus of the stria terminalis and lateral habenula receive projections from the MR but the nucleus accumbens and caudate do not13. This may be important since rats with a lesion in the MR show a potentiation of morphine analgesia or a reduction in the amount of pain produced in the formalin test1, 3. The participation of forebrain structures in morphine analgesia in formalin test pain does not mean that brainstem structures play no role in this analgesia. Possibly the forebrain structures produce their effects through action on brainstem structures, which in turn, as discussed in the introduction with respect to brief, rapidly rising pain, may act to inhibit incoming pain signals at the spinal level. However, in these pain tests supraspinal structures are thought to send efferents through the DLF to inhibit pain signals at the spinal level8d2, 20.23. Yet destruction of the DLF has no effect on morphine analgesia in the formalin test (L. Watkins, personal communication, 1983). Therefore, if brainstem structures are important for morphine analgesia in the formalin test, they play a role different from that in brief, rapidly rising pain. Alternatively, the analgesia produced by morphine's action on forebrain structures may not depend on a descending system at all. The morphine may act at forebrain levels to block or alter incoming nociceptive signals so that they are no longer perceived as painful. Amodei and PaxinosS, using unilateral knife cuts through the medial forebrain bundle and the medial internal capsule and/or the thalamus, have already provided evidence that nociceptive signals produced by a formalin injection are processed in the ipsilateral forebrain. It is possible that i.c.v. morphine relieves pain in the formalin test in a manner similar to that in which frontal lobotomy or cingu-
285 lotomy relieve chronic pain in humans. Both frontal lobotomy and cingulotomy appear to leave the sensory aspect of pain intact, but greatly reduce the anxiety and suffering that accompany chronic pain 18,21. In the foot-flick test, morphine produced bilateral analgesia. This does not mean that the neural mechanism through which morphine produces its analgesic effect in the foot-flick test has no somatotopic organization with respect to laterality. The results simply indicate that, with this method of morphine administration, no lateralization of effect is present. It is noteworthy that Yaksh et al. 41 report analgesia briefly restricted to the ipsilateral face and forepaw after injection of morphine into the rostral PAG using the pinch test. Very high doses of morphine were required to produce an increase in foot-flick latency. Pilot studies conducted with low doses, similar to those used in the formalin test, indicated that they did not affect the foot-flick latency. It can be seen in Fig. 3 that even 25 #g of morphine had no effect on foot-flick latency. Yet the foot-flick test is sensitive to the analgesic effect of systemic morphine. Studies in this laboratory have shown that 12 mg/kg of morphine produce a sizeable increase in latency to withdraw the paw from hot water. Using focused light as a source of heat, Levine et al. 27 report that the foot-flick is similar to the tail-flick and responds similarly to systemic morphine. If the structures responsible for morphine analgesia in tests measuring brief, rapidly rising pain are in the brainstem, near the third and fourth ventricles and the aqueduct of Sylvius, as has been suggested by others a°,2°,3°, then it is possible that very high doses of morphine are needed in the foot-flick test to enable a sufficient amount of morphine to reach these brainstem structures. This suggestion is supported by the fact that analgesia was increased by simply increasing the volume of the microinjection, which would be expected to increase the diffusion of the morphine from the injection site. In fact, Herz et al. 24, measuring the latency of a licking reaction produced by stimulating the tooth-pulp in the rabbit, report that i.c.v, morphine restricted to the lateral and third ventricles produces no analgesia, but morphine restricted to the aqueduct and fourth ventricle produces analgesia. In addition, Van Ree 40, using the hot plate test in rats, reports that when the i.c.v, injection is directed at the lateral or third ventricles, 4 times more morphine is required to produce analge-
sia than when the morphine is injected into the fourth ventricle. There is another indication that morphine did not act, in the foot-flick test, on structures close to the lateral ventricle. With doses of 50/2g and 100 pg of morphine, the analgesia did not reach a maximum until 30-45 min after i.c.v, injection, suggesting that the morphine had to diffuse from the lateral ventricle before it could produce its maximal effect. With the 200 pg dose, the maximal effect was attained 15 min after injection, at the first time of testing. This may reflect the arbitrary ceiling on paw withdrawal latency, since at this dose and time of measurement there were several rats that left their paws in the water for the maximal 12 s. Herz et al. 24 using tooth-pulp stimulation in the rabbit, report that the maximal analgesic effect is reached more quickly after injection of morphine into the aqueduct than into the lateral ventricle. In conclusion, the data reveal that the system through which i.c.v, morphine exerts its analgesic effect on continuous, moderate pain is organized so that each hemisphere modulates pain in the ipsilateral but not the contralateral hindpaw. Although it is clear that a considerable degree of lateralization exists, it is possible that each hemisphere also exerts minor control over continuous, moderate pain in the contralateral hindpaw. Possibly, this lateralization applies to the entire body, but further experimentation is necessary to demonstrate this. No evidence was found for a similar somatotopic organization for modulation of brief, rapidly rising pain. Furthermore, the data strongly suggest that forebrain structures near the lateral ventricle participate in morphine analgesia for continuous, moderate pain. The data also suggest that the neural structures responsible for morphine analgesia in brief, rapidly rising pain are different from those responsible for morphine's effect on continuous, moderate pain. ACKNOWLEDGEMENTS The results of this study were published in abstract form in Society for Neuroscience Abstracts, 1983. This research was supported by Natural Sciences and Engineering Research Council of Canada Grant A7891 to Dr. Ronald Melzack. S. R. Cohen is supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship.
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