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Alterations in nociceptirve threshold and morphine-induced analgesia produced by intrathecally administered amine antagonists
Brain Research, 218 (1981) 393-399 Elsevier/North-Holland Biomedical Press
Alterations
in nociceptive
393
threshold
duced by intrathecally
administered
HERBERT
and DONNA
K. PROUDFIT
and morphine-induced
analgesia
pro-
amine antagonists
L. HAMMOND
Department of Pharmacology, University of Illinois at the Medical Center, Chicago, Ill. 60612 and Neurosurgical Research, Mayo Foundation, Rochester, Minn. 55901 (U.S.A.)
(Accepted March 23rd, 1981) Key words: morphine
intrathecal
-
phentolamine injection
-
methysergide
-
nociceptive threshold
-
analgesia -
Intrathecal administration of either methysergide or phentolamine produced hyperalgesia. This suggests that tonically active serotonergic and noradrenergic neuronal systems modulate sensitivity to nociceptive stimuli at the level of the spinal cord. Methysergide did not attenuate the analgesia induced by either 2.0 or 7.5 mg/kg morphine (s.c.), while phentolamine attenuated the analgesia induced by 2.0, but not 7.5 mg/kg morphine. These findings suggest that bulbospinal serotonergic neurons are not integral components of the neuronal circuitry which mediates opiate-induced analgesia. Noradrenergic neurons, however, appear to mediate a portion of such analgesia.
Numerous studies have provided evidence for the proposal that the analgetic action of systemically-administered opiates is mediated by raphe-spinal serotonergic neuronsr$s*6,1*and spinally-projecting noradrenergic neuronsrsJ6. The present studies were designed to test this hypothesis directly, by examining the capacity of intrathecally-administered methysergide, a serotonergic antagonist, and phentolamine, a noradrenergic antagonist, to attenuate the analgesia induced by systemically-administered morphine. A preliminary report of these findings has been presented elsewhere2. Male rats (Sprague-Dawley derived) weighing 425-450 g were implanted under ether anesthesia with an intrathecal catheter using a modified method of Yaksh and RudyIT. Animals were housed individually with free access to food and water; 7 days were allowed to elapse prior to analgesiometric testing. Those animals which sustained motor or sensory deficits from implantation of the catheter were discarded from the study. The response threshold to noxious thermal stimuli was determined for all animals using both the tail-flick (TF) and the hot-plate (HP) tests. The TF determination was made first and was followed immediately by the HP test. The time which elapsed prior to reflex removal of the tail from the beam of a high intensity light was termed the tail-flick latency (TFL). The average of 3 successive determinations was recorded. Hot-plate latency (HPL) was defined as the time elapsed to a lick of the hind paw or a jump after placement on a 55 “C copper hot plate. One measurement of HPL was made. Animals which failed to execute the appropriate end-point response OOOS-8993/81/0000-0000/$02.50
0
Elsevier/North-Holland
Biomedical Press
394
by 14 set on the TF or by 40 set on the HP were removed and assigned the cut-oh latency. All drug solutions were freshly made on the day of testing. Morphine sulfate (Merck) was dissolved in physiological saline (0.9 “/, NaCl) while phentolamine hydrochloride (Ciba-Geigy) and methysergide bimaleate (Sandoz) were dissolved in physiological saline, passed through a 0.2 pm Millipore filter, and adjusted to pH 7.0 prior to intrathecal injection. Thirty lug of either agent in 15 ,~l saline was injected into the spinal subarachnoid space and followed by 10 ~1 of saline to ensure delivery of the antagonists into the intrathecal space. Sixty-three rats were randomly assigned to 9 groups of 7 rats each. The capacity of intrathecally-administered methysergide or phentolamine to alter the analgesia produced by systemically administered morphine was investigated. Following determination of baseline nociceptive threshold, animals in 3 groups each received 2.0 mg/kg morphine sulfate (s.c.). Thirty minutes later (time of peak effect) the elevation of nociceptive threshold produced by morphine was assessed. Immediately following this assessment of analgesia, the following drugs were injected intrathecally : group I,30 pg methysergide; group 2, 30 ,ug phentolamine; and group 3, saline. Nociceptive threshold was then re-assessed 5, 15 and 30 min later. The alterations in morphine-induced analgesia produced by intrathecal injection of methysergide or of phentolamine were compared with those produced by intrathecal injection of saline by means of a twoway analysis of variance with repeated measuresa. Individual comparisons were made using the Newman-Keuls testa. Baseline nociceptive threshold was determined for 3 additional groups of animals and each received 7.5 mg/kg morphine (s.c.). Thirty minutes later, the elevation of nociceptive threshold was assessed. Immediately following this assessment of morphine-induced analgesia, the following drugs were injected intrathecally : group 4,30 pg methysergide; group 5,30 ,ug phentolamine; and group 6, saline. A two-way analysis of variance with repeated measures was used to compare the alterations in morphine-induced analgesia produced by intrathecal injection of methysergide or phentolamine with those produced by intrathecal injection of saline. Three additional groups were used to assess the capacity of intrathecally-administered phentolamine, methysergide or saline given alone to alter nociceptive threshold. Following determination of baseline nociceptive threshold, these 3 groups of animals received physiological saline (0.1 ml/100 g b. wt.; s.c.). Thirty minutes later, the alteration in nociceptive threshold produced by saline was assessed. Immediately after this assessment, the following drugs were injected intrathecally : group 7, 30 pg methysergide; group 8, 30 ,ug phentolamine; and group 9, saline. The alterations in nociceptive threshold produced by intrathecal administration of methysergide or phentolamine were compared with those produced by intrathecal injection of saline by means of a two-way analysis of variance a. Newman-Keuls tests was used to make comparisons between individual group means. The capacity of intrathecally-administered methysergide or phentolamine to alter nociceptive threshold was assessed in 3 groups of animals. These 3 groups of animals all had similar preinjection baseline thresholds since there were no statistically significant differences (Newman-Keuls test, P > 0.05) in mean TFLs or HPLs
395
54 o+
df Pro
0 (A)
I 30
A
I 40
I 50
I 65
(6) MINUTES
Fig. 1. Effect of intrathecally-administered saline, phentolamine and methysergide on nociceptive threshold assessed using the tail-flick test (upper graph) and the hot-plate test (lower graph). Values represent mean h S.E.M. response latencies. Preinjection baseline response latencies are indicated by the abbreviation ‘Pre’ on the abscissa. The letter A indicates the time at which each of the 3 groups was injected with saline (s.c.). Response latencies were measured 30 min after the saline injection. The arrow at B indicates the time at which either saline (filled circles), phentolamine (open squares), or methysergide (open triangles) was injected intrathecally.
between any of these groups (Fig. 1). In addition, no statistically significant differences in either mean TFL or HPL were observed between any of these three groups 30 min after subcutaneous saline (Newman-Keuls test, P > 0.05). However, the mean TFL of the phentolamine group was elevated 30 min after subcutaneous administration of saline when compared with the mean preinjection baseline latency for this group (Newman-Keuls test, P < 0.05). The subsequent intrathecal injection of saline (group 9) did not significantly alter HPL at any time (two-way ANOVA, P > 0.05). In contrast, the intrathecal administration of either methysergide (group 7) or phentolamine (group 8) significantly decreased mean HPLs as compared to that of group 9 which had received saline intrathecally (two-way ANOVA, P < 0.05)(Fig. 1). The reductions in mean HPLs produced by intrathecal injection of both phentolamine and methysergide were significantly different (Newman-Keuls test, P < 0.05) from that of the saline-injected group at all 3 time points (5, 15 and 30 min postinjection). Intrathecal administration of saline did not significantly alter the mean TFL (two-way ANOVA, P > 0.05). In contrast, both phentolamine and methysergide significantly decreased the mean TFL within 5 min after intrathecal injection as compared to the group which had received saline intrathecally (Newman-Keuls test, P < 0.05) (Fig. 1). Mean TFLs for both groups remained depressed 15 min after the intrathecal injection of either antagonist. The phentolamine group had recovered by 30 min, since the mean TFL at 30 min was not significantly different from that for the saline-injected group
396
Ok
,” / 30, (A)
A
I
I
40
50
I
65
(8) MINUTES
Fig. 2. Effect of intrathecally-administered saline, phentolamine and methysergide on the analgesia induced by morphine sulfate (2 mg/kg, s.c.). Values represent mean i S.E.M. response latencies. Preinjection baseline response latencies are indicated by the abbreviation ‘Pre’ on the abscissa. The letter A indicates the time at which each of the 3 groups was injected with morphine sulfate. Response latencies were measured 30 min after the morphine injection. The arrow at B indicates the time at which either saline (filled circles), phentolamine (open squares), or methysergide (open triangles) was injected intrathecally.
(Newman-Keuls test, P > 0.05). However, the mean TFL for the methysergide group was still significantly lower than that for the saline group at 30 min (Newman-Keuls test, P < 0.05). The capacity of methysergide or phentolamine, when injected intrathecally, to attenuate the analgesia induced by either 2.0 or 7.5 mg/kg of morphine was assessed. Both doses of morphine produced a significant elevation of both TFL and HPL, although 7.5 mg/kg of morphine elevated these parameters to a greater extent than did the 2.0 mg/kg dose (two-way ANOVA, P < 0.05). The extent to which 2.0 mg/kg of morphine elevated either TFL or HPL did not differ between any of the 3 groups (twoway ANOVA, P > 0.05). Similarly, the increase in either TFL or H,PL produced by the 7.5 mg/kg dose of morphine did not differ among the 3 groups (two-way ANOVA, P > 0.05). The subsequent intrathecal injection of saline did not attenuate the elevation of HPL induced by either dose of morphine. Intrathecal injection of methysergide also failed to attenuate the analgesia induced by either 2.0 or 7.5 mg/kg of morphine when assessed using the hot-plate test. Instead, intrathecally-administered methysergide appeared to augment the analgesia produced by 2.0 mg/kg morphine since the mean HPLs at 5, 15 and 30 min after methysergide were significantly greater than the mean HPL for this group determined 30 min after systemic morphine (Newman-Keuls test, P (: 0.05). However, there were no significant differences between the mean HPLs
391
for the control group and those for the methysergide group at any time point (NewmanKeuls test, P > 0.05). No statistically significant alterations in HPL were observed when methysergide was injected following the 7.5 mg/kg dose of morphine (Newman-Keuls test, P > 0.05). A significant attenuation of morphine-induced analgesia was produced, however, by the intrathecal injection of phentolamine as compared to the group which had received saline (two-way ANOVA, P -c 0.05) (Fig. 2). This attenuation was surmountable since no attenuation of the analgesia induced by 7.5 mg/kg morphine was observed following intrathecally-administered phentolamine (Table I). When analgesia was measured using the TF test, no attenuation of the analgesia induced by either 2.0 or 7.5 mg/kg morphine was observed following the intrathecal administration of either saline, methysergide, or phentolamine (two-way ANOVA, P > 0.05) (Fig. 2; Table I). A major finding of this study was the ability of either amine antagonist to decrease nociceptive threshold. Methysergide produced a pronounced hyperalgesia following its injection into the intrathecal space. This hyperalgesia occurred within 5 min, persisted for at least 30 min, and was evident using both analgesiometric tests. Phentolamine produced identical effects with one exception; the hyperalgesia was no longer evident by 30 min using the tail flick test. Hyperalgesia has similarly been observed following the depletion of either serotonin or norepinephrine in the spinal cord by intrathecal injection of 5,6_dihydroxytryptamine or 6-hydroxydopamine, respectivelyi’. Conversely, intrathecal injection of either serotonini5,1g, or norepinephrine4Jz has been reported to produce analgesia. Together, these findings are consistent with the concept of tonically active serotonergic and noradrenergic neuronal systems which gate the transmission of nociceptive impulses at the level of the spinal cord. The tonically active serotonergic system probably originates in the caudal raphe nuclei which contain spinally-projecting serotonergic neurons. The appearance of hyperalgesia following lesions of the caudal raphe nuclei7sJ0 which TABLE I Effect of intrathecally-administered saline, phentolamine or methysergide ally-administered morphine (7.5 mglkg) to induce antinociception
on the capacity
of systemic-
Values represent mean response latencies & S.E.M. in sec. Analgesiometric test
Intrathecal drug
Tail-flick
Saline Phentolamine Methysergide
Hot-plate
Saline Phentolamine Methysergide
Baseline * latency
30 min after morphine
Time after intrathecal injection (min) ___5 15 30
3.1 f 0.2 3.1 f 0.2 3.6 f 0.2
14.0 i 0.0 14.0 5 0.0 14.0 rt 0.0
14.0 i 0.0 14.0 f 0.0 14.0 5 0.0
14.0 + 0.0 14.0 + 0.0 14.0 f 0.0
13.7 f 0.0 14.0 i_ 0.0 14.0 i 0.0
13.3 -i- 1.6 12.8 + 1.2 13.1 i 1.1
30.3 f 4.1 31.1 f 4.3 35.9 f 1.6
29.3 & 4.6 27.4 zt 5.6 38.4 i 1.6
32.4 It 3.8 32.2 f 4.5 39.2 & 0.8
35.7 i 3.0 35.6 & 3.3 39.9 & 0.1
* These values represent response fatencies determined immediately before the injection of morphine.
398 decrease the spinal cord content more, inactivation anesthetic
of serotonins
of raphe-spinal
in the raphe magnus
The analgesia thecal injection
produced
neurons
lends support produced
also induces antagonist
was not attenuated
methysergide
employed
inadequate,
hyperalgesia
however, it did produce
the analgesia
an effect; a significant
tests. Also, 30 ,ug of methysergide
produced
by the intrathecal
injection
by intra-
when the tail-flick test was
used. It may be argued that the 30 lug dose of methysergide two analgesiometric
Furterof a local
hyperalgesiaY.
by either dose of morphine
of the serotonergic
to this hypothesis.
by the microinjection
in this study was as assessed by
has been shown to antagonize
of as much as 200 lug of serotoninl”.
The present findings and those of our previous studyii who also failed to demonstrate a significant attenuation
concur with those of Vogt’* of morphine-induced analge-
sia after the administration
(i.c.v.) which depleted
cord serotonin
by 90%.
of 5,7_dihydroxytryptamine Thus,
it would
appear
that the raphe-spinal
spinal
serotonergic
neurons are not required for the expression of the analgesia produced by systemicallyadministered morphine. Although methysergide did not alter morphine analgesia using the tail flick test, when the hot plate test was used methysergide produced morphine-induced analgesia. However, the potentiation only when methysergide
was given following
an apparent potentiation of was statistically significant
the 2.0 mg/kg dose of morphine,
the 7.5 mg/kg dose. This potentiation is most likely a result of repeated hot-plate since the saline injected controls also exhibited a tendency prolonged replication
but not
testing on the toward more
hot-plate latencies with repeated testing (see Figs. 1 and 2). Furthermore, in a of these studies for another purpose hot-plate latencies were also elevated,
but were not statistically
significant,
following
methysergide
given intrathecally
after
doses of 3, 5 and 7.5 mg/kg of morphine using 34 rats. Intrathecal injection of the noradrenergic antagonist phentolamine attenuated the analgesia induced by 2.0 mg/kg of morphine. However, this attenuation was apparent only when analgesia attenuation was surmountable,
was assessed using the hot-plate test. Furthermore, this since phentolamine was unable to antagonize the more
potent analgesia produced by 7.5 mg/kg of morphine. This observation is consistent with our previous report11 which demonstrated that depletion of spinal cord norepinephrine content produced a modest reduction in the capacity of morphine to induce analgesia using the hot-plate, but not the tail-flick test. It is possible that the reduction in morphine analgesia produced by intrathecal phentolamine is a result of the hyperalgesia produced by phentolamine (see Fig. 1). However, this proposal does not seem likely, since analgesia induced by the injection of morphine is not attenuated by the induction of hyperalgesia produced by either the injection of local anesthetics in the nucleus raphe magnuss or depletion of spinal cord serotonin contentl. These findings are consistent with other reports suggesting that noradrenergic neurons participate in mediating opiate analgesiars,is. However, since intrathecally-injected phentolamine was ineffective in altering morphine-induced analgesia using the tail-flick test and only moderately effective using the hot-plate test, it appears that bulbospinal noradrenergic neurons are of minor importance in mediating opiate-induced analgesia. The results of the present study suggest that: (1) tonically active serotonergic and
399 noradrenergic neuronal systems regulate the transmission of nociceptive information at the level of the spinal cord; (2) serotonergic neurons which project to the spinal cord do not participate in the mediation of analgesia induced by systemically administered morphine to an appreciable extent; and (3) noradrenergic neuronal systems which terminate in the spinal cord may mediate a portion of the analgetic action of opiates. We would like to acknowledge the skillful assistance of Ms. Joann Hettasch. This study was supported by Grant NS 12649 to H.K.P.
1 Fields, H. L. and Basbaum, A. I., Brainstem control of spinal pain-transmission neurons, Atin. Rev. Physiol., 40 (1978) 217-248. 2 Hammond, D. L. and Proudfit, H. K., Effects of intrathecally administered amine antagonists on nociceptive threshold and morphine-induced analgesia, Neurosci. Abstr., 6 (1980) 433. 3 Keppel, G., Design and Analysis: A Researcher’s Handbook, Prentice-Hall, New Jersey, 1973. 4 Kuraishi, Y., Harada, Y. and Takagi, H., Noradrenaline regulation of pain-transmission in the Brain Research, 174 (1979) 333-336. spinal cord mediated by a-adrenoreceptors, 5 Liebeskind, J. C., Mayer, D. J. and Akil, H., Central mechanisms of pain inhibition: Studies of analgesia from focal brain stimulation, Advanc. Neural., 4 (1974) 261-269. 6 Mayer, D. J. and Price, D. D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 3799404. 7 Proudfit, H. K., Time-course of alterations in morphine-induced analgesia and nociceptive threshold following medullary raphe lesions, Neuroscience, in press. 8 Proudfit, H. K., Effects of raphe magnus and raphe pallidus lesions on morphine-induced analgesia and spinal cord monoamines, Pharmacol. Biochem. Behav., 13 (1980) 705-714. 9 Proudfit, H. K., Reversible inactivation of raphe magnus neurons: effect on nociceptive threshold and morphine-induced analgesia, Brain Research, 201 (1980) 459464. IO Proudfit, H. K. and Anderson, E. G., Morphine analgesia: blockade by raphe magnus lesions, Brain Research, 98 (1975) 612-618. I1 Proudfit, H. K. and Yaksh, T. L., Nociceptive threshold and morphine analgesia: alterations following the intrathecal administration of 6-hydroxydopamine and 5,6-dihydroxytryptamine, Neurosci. Abstr., 6 (1980) 433. 12 Reddy, S. V. R., Maderdrut, J. L. and Yaksh, T. L., Spinal cord pharmacology agonist-mediated antinociception, J. Pharmacol. exp. Ther., 213 (1980) 525-533.
of adrenergic
13 Shiomi, H. and Takagi, H., Morphine analgesia and the bulbospinal noradrenergic system: increase in the concentration of normetanephrine in the spinal cord of the rat caused by analgesics, Brit. J. Pharmacol., 52 (1974) 519-526. 14 Vogt, M., The effect of lowering the 5-hydroxytryptamine content of the rat spinal cord on analgesia produced by morphine, J. Physiol. (Land.), 236 (1974) 483498. 15 Wang, J. K., Antinociceptive effect of intrathecally administered serotonin, Anesthesiology, 47 (1977) 269-271.
16 Yaksh, T. L., Direct evidence that spinal serotonin and noradrenaline terminals mediate the spinal antinociceptive effects of morphine in the periaqueductal gray, Brain Research, 160 (1979) 180-185. 17 Yaksh, T. L. and Rudy, T. A.,, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav., 197 (1976) 1031-1036. 18 Yaksh, T. L. and Rudy, T. A., Narcotic analgetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 259-299. 19 Yaksh, T. L. and Wilson, P. R., Spinal serotonin terminal system mediates antinociception, J. Pharmacol. exp. Ther., 208 (1979) 446453.
Alterations
in nociceptive
393
threshold
duced by intrathecally
administered
HERBERT
and DONNA
K. PROUDFIT
and morphine-induced
analgesia
pro-
amine antagonists
L. HAMMOND
Department of Pharmacology, University of Illinois at the Medical Center, Chicago, Ill. 60612 and Neurosurgical Research, Mayo Foundation, Rochester, Minn. 55901 (U.S.A.)
(Accepted March 23rd, 1981) Key words: morphine
intrathecal
-
phentolamine injection
-
methysergide
-
nociceptive threshold
-
analgesia -
Intrathecal administration of either methysergide or phentolamine produced hyperalgesia. This suggests that tonically active serotonergic and noradrenergic neuronal systems modulate sensitivity to nociceptive stimuli at the level of the spinal cord. Methysergide did not attenuate the analgesia induced by either 2.0 or 7.5 mg/kg morphine (s.c.), while phentolamine attenuated the analgesia induced by 2.0, but not 7.5 mg/kg morphine. These findings suggest that bulbospinal serotonergic neurons are not integral components of the neuronal circuitry which mediates opiate-induced analgesia. Noradrenergic neurons, however, appear to mediate a portion of such analgesia.
Numerous studies have provided evidence for the proposal that the analgetic action of systemically-administered opiates is mediated by raphe-spinal serotonergic neuronsr$s*6,1*and spinally-projecting noradrenergic neuronsrsJ6. The present studies were designed to test this hypothesis directly, by examining the capacity of intrathecally-administered methysergide, a serotonergic antagonist, and phentolamine, a noradrenergic antagonist, to attenuate the analgesia induced by systemically-administered morphine. A preliminary report of these findings has been presented elsewhere2. Male rats (Sprague-Dawley derived) weighing 425-450 g were implanted under ether anesthesia with an intrathecal catheter using a modified method of Yaksh and RudyIT. Animals were housed individually with free access to food and water; 7 days were allowed to elapse prior to analgesiometric testing. Those animals which sustained motor or sensory deficits from implantation of the catheter were discarded from the study. The response threshold to noxious thermal stimuli was determined for all animals using both the tail-flick (TF) and the hot-plate (HP) tests. The TF determination was made first and was followed immediately by the HP test. The time which elapsed prior to reflex removal of the tail from the beam of a high intensity light was termed the tail-flick latency (TFL). The average of 3 successive determinations was recorded. Hot-plate latency (HPL) was defined as the time elapsed to a lick of the hind paw or a jump after placement on a 55 “C copper hot plate. One measurement of HPL was made. Animals which failed to execute the appropriate end-point response OOOS-8993/81/0000-0000/$02.50
0
Elsevier/North-Holland
Biomedical Press
394
by 14 set on the TF or by 40 set on the HP were removed and assigned the cut-oh latency. All drug solutions were freshly made on the day of testing. Morphine sulfate (Merck) was dissolved in physiological saline (0.9 “/, NaCl) while phentolamine hydrochloride (Ciba-Geigy) and methysergide bimaleate (Sandoz) were dissolved in physiological saline, passed through a 0.2 pm Millipore filter, and adjusted to pH 7.0 prior to intrathecal injection. Thirty lug of either agent in 15 ,~l saline was injected into the spinal subarachnoid space and followed by 10 ~1 of saline to ensure delivery of the antagonists into the intrathecal space. Sixty-three rats were randomly assigned to 9 groups of 7 rats each. The capacity of intrathecally-administered methysergide or phentolamine to alter the analgesia produced by systemically administered morphine was investigated. Following determination of baseline nociceptive threshold, animals in 3 groups each received 2.0 mg/kg morphine sulfate (s.c.). Thirty minutes later (time of peak effect) the elevation of nociceptive threshold produced by morphine was assessed. Immediately following this assessment of analgesia, the following drugs were injected intrathecally : group I,30 pg methysergide; group 2, 30 ,ug phentolamine; and group 3, saline. Nociceptive threshold was then re-assessed 5, 15 and 30 min later. The alterations in morphine-induced analgesia produced by intrathecal injection of methysergide or of phentolamine were compared with those produced by intrathecal injection of saline by means of a twoway analysis of variance with repeated measuresa. Individual comparisons were made using the Newman-Keuls testa. Baseline nociceptive threshold was determined for 3 additional groups of animals and each received 7.5 mg/kg morphine (s.c.). Thirty minutes later, the elevation of nociceptive threshold was assessed. Immediately following this assessment of morphine-induced analgesia, the following drugs were injected intrathecally : group 4,30 pg methysergide; group 5,30 ,ug phentolamine; and group 6, saline. A two-way analysis of variance with repeated measures was used to compare the alterations in morphine-induced analgesia produced by intrathecal injection of methysergide or phentolamine with those produced by intrathecal injection of saline. Three additional groups were used to assess the capacity of intrathecally-administered phentolamine, methysergide or saline given alone to alter nociceptive threshold. Following determination of baseline nociceptive threshold, these 3 groups of animals received physiological saline (0.1 ml/100 g b. wt.; s.c.). Thirty minutes later, the alteration in nociceptive threshold produced by saline was assessed. Immediately after this assessment, the following drugs were injected intrathecally : group 7, 30 pg methysergide; group 8, 30 ,ug phentolamine; and group 9, saline. The alterations in nociceptive threshold produced by intrathecal administration of methysergide or phentolamine were compared with those produced by intrathecal injection of saline by means of a two-way analysis of variance a. Newman-Keuls tests was used to make comparisons between individual group means. The capacity of intrathecally-administered methysergide or phentolamine to alter nociceptive threshold was assessed in 3 groups of animals. These 3 groups of animals all had similar preinjection baseline thresholds since there were no statistically significant differences (Newman-Keuls test, P > 0.05) in mean TFLs or HPLs
395
54 o+
df Pro
0 (A)
I 30
A
I 40
I 50
I 65
(6) MINUTES
Fig. 1. Effect of intrathecally-administered saline, phentolamine and methysergide on nociceptive threshold assessed using the tail-flick test (upper graph) and the hot-plate test (lower graph). Values represent mean h S.E.M. response latencies. Preinjection baseline response latencies are indicated by the abbreviation ‘Pre’ on the abscissa. The letter A indicates the time at which each of the 3 groups was injected with saline (s.c.). Response latencies were measured 30 min after the saline injection. The arrow at B indicates the time at which either saline (filled circles), phentolamine (open squares), or methysergide (open triangles) was injected intrathecally.
between any of these groups (Fig. 1). In addition, no statistically significant differences in either mean TFL or HPL were observed between any of these three groups 30 min after subcutaneous saline (Newman-Keuls test, P > 0.05). However, the mean TFL of the phentolamine group was elevated 30 min after subcutaneous administration of saline when compared with the mean preinjection baseline latency for this group (Newman-Keuls test, P < 0.05). The subsequent intrathecal injection of saline (group 9) did not significantly alter HPL at any time (two-way ANOVA, P > 0.05). In contrast, the intrathecal administration of either methysergide (group 7) or phentolamine (group 8) significantly decreased mean HPLs as compared to that of group 9 which had received saline intrathecally (two-way ANOVA, P < 0.05)(Fig. 1). The reductions in mean HPLs produced by intrathecal injection of both phentolamine and methysergide were significantly different (Newman-Keuls test, P < 0.05) from that of the saline-injected group at all 3 time points (5, 15 and 30 min postinjection). Intrathecal administration of saline did not significantly alter the mean TFL (two-way ANOVA, P > 0.05). In contrast, both phentolamine and methysergide significantly decreased the mean TFL within 5 min after intrathecal injection as compared to the group which had received saline intrathecally (Newman-Keuls test, P < 0.05) (Fig. 1). Mean TFLs for both groups remained depressed 15 min after the intrathecal injection of either antagonist. The phentolamine group had recovered by 30 min, since the mean TFL at 30 min was not significantly different from that for the saline-injected group
396
Ok
,” / 30, (A)
A
I
I
40
50
I
65
(8) MINUTES
Fig. 2. Effect of intrathecally-administered saline, phentolamine and methysergide on the analgesia induced by morphine sulfate (2 mg/kg, s.c.). Values represent mean i S.E.M. response latencies. Preinjection baseline response latencies are indicated by the abbreviation ‘Pre’ on the abscissa. The letter A indicates the time at which each of the 3 groups was injected with morphine sulfate. Response latencies were measured 30 min after the morphine injection. The arrow at B indicates the time at which either saline (filled circles), phentolamine (open squares), or methysergide (open triangles) was injected intrathecally.
(Newman-Keuls test, P > 0.05). However, the mean TFL for the methysergide group was still significantly lower than that for the saline group at 30 min (Newman-Keuls test, P < 0.05). The capacity of methysergide or phentolamine, when injected intrathecally, to attenuate the analgesia induced by either 2.0 or 7.5 mg/kg of morphine was assessed. Both doses of morphine produced a significant elevation of both TFL and HPL, although 7.5 mg/kg of morphine elevated these parameters to a greater extent than did the 2.0 mg/kg dose (two-way ANOVA, P < 0.05). The extent to which 2.0 mg/kg of morphine elevated either TFL or HPL did not differ between any of the 3 groups (twoway ANOVA, P > 0.05). Similarly, the increase in either TFL or H,PL produced by the 7.5 mg/kg dose of morphine did not differ among the 3 groups (two-way ANOVA, P > 0.05). The subsequent intrathecal injection of saline did not attenuate the elevation of HPL induced by either dose of morphine. Intrathecal injection of methysergide also failed to attenuate the analgesia induced by either 2.0 or 7.5 mg/kg of morphine when assessed using the hot-plate test. Instead, intrathecally-administered methysergide appeared to augment the analgesia produced by 2.0 mg/kg morphine since the mean HPLs at 5, 15 and 30 min after methysergide were significantly greater than the mean HPL for this group determined 30 min after systemic morphine (Newman-Keuls test, P (: 0.05). However, there were no significant differences between the mean HPLs
391
for the control group and those for the methysergide group at any time point (NewmanKeuls test, P > 0.05). No statistically significant alterations in HPL were observed when methysergide was injected following the 7.5 mg/kg dose of morphine (Newman-Keuls test, P > 0.05). A significant attenuation of morphine-induced analgesia was produced, however, by the intrathecal injection of phentolamine as compared to the group which had received saline (two-way ANOVA, P -c 0.05) (Fig. 2). This attenuation was surmountable since no attenuation of the analgesia induced by 7.5 mg/kg morphine was observed following intrathecally-administered phentolamine (Table I). When analgesia was measured using the TF test, no attenuation of the analgesia induced by either 2.0 or 7.5 mg/kg morphine was observed following the intrathecal administration of either saline, methysergide, or phentolamine (two-way ANOVA, P > 0.05) (Fig. 2; Table I). A major finding of this study was the ability of either amine antagonist to decrease nociceptive threshold. Methysergide produced a pronounced hyperalgesia following its injection into the intrathecal space. This hyperalgesia occurred within 5 min, persisted for at least 30 min, and was evident using both analgesiometric tests. Phentolamine produced identical effects with one exception; the hyperalgesia was no longer evident by 30 min using the tail flick test. Hyperalgesia has similarly been observed following the depletion of either serotonin or norepinephrine in the spinal cord by intrathecal injection of 5,6_dihydroxytryptamine or 6-hydroxydopamine, respectivelyi’. Conversely, intrathecal injection of either serotonini5,1g, or norepinephrine4Jz has been reported to produce analgesia. Together, these findings are consistent with the concept of tonically active serotonergic and noradrenergic neuronal systems which gate the transmission of nociceptive impulses at the level of the spinal cord. The tonically active serotonergic system probably originates in the caudal raphe nuclei which contain spinally-projecting serotonergic neurons. The appearance of hyperalgesia following lesions of the caudal raphe nuclei7sJ0 which TABLE I Effect of intrathecally-administered saline, phentolamine or methysergide ally-administered morphine (7.5 mglkg) to induce antinociception
on the capacity
of systemic-
Values represent mean response latencies & S.E.M. in sec. Analgesiometric test
Intrathecal drug
Tail-flick
Saline Phentolamine Methysergide
Hot-plate
Saline Phentolamine Methysergide
Baseline * latency
30 min after morphine
Time after intrathecal injection (min) ___5 15 30
3.1 f 0.2 3.1 f 0.2 3.6 f 0.2
14.0 i 0.0 14.0 5 0.0 14.0 rt 0.0
14.0 i 0.0 14.0 f 0.0 14.0 5 0.0
14.0 + 0.0 14.0 + 0.0 14.0 f 0.0
13.7 f 0.0 14.0 i_ 0.0 14.0 i 0.0
13.3 -i- 1.6 12.8 + 1.2 13.1 i 1.1
30.3 f 4.1 31.1 f 4.3 35.9 f 1.6
29.3 & 4.6 27.4 zt 5.6 38.4 i 1.6
32.4 It 3.8 32.2 f 4.5 39.2 & 0.8
35.7 i 3.0 35.6 & 3.3 39.9 & 0.1
* These values represent response fatencies determined immediately before the injection of morphine.
398 decrease the spinal cord content more, inactivation anesthetic
of serotonins
of raphe-spinal
in the raphe magnus
The analgesia thecal injection
produced
neurons
lends support produced
also induces antagonist
was not attenuated
methysergide
employed
inadequate,
hyperalgesia
however, it did produce
the analgesia
an effect; a significant
tests. Also, 30 ,ug of methysergide
produced
by the intrathecal
injection
by intra-
when the tail-flick test was
used. It may be argued that the 30 lug dose of methysergide two analgesiometric
Furterof a local
hyperalgesiaY.
by either dose of morphine
of the serotonergic
to this hypothesis.
by the microinjection
in this study was as assessed by
has been shown to antagonize
of as much as 200 lug of serotoninl”.
The present findings and those of our previous studyii who also failed to demonstrate a significant attenuation
concur with those of Vogt’* of morphine-induced analge-
sia after the administration
(i.c.v.) which depleted
cord serotonin
by 90%.
of 5,7_dihydroxytryptamine Thus,
it would
appear
that the raphe-spinal
spinal
serotonergic
neurons are not required for the expression of the analgesia produced by systemicallyadministered morphine. Although methysergide did not alter morphine analgesia using the tail flick test, when the hot plate test was used methysergide produced morphine-induced analgesia. However, the potentiation only when methysergide
was given following
an apparent potentiation of was statistically significant
the 2.0 mg/kg dose of morphine,
the 7.5 mg/kg dose. This potentiation is most likely a result of repeated hot-plate since the saline injected controls also exhibited a tendency prolonged replication
but not
testing on the toward more
hot-plate latencies with repeated testing (see Figs. 1 and 2). Furthermore, in a of these studies for another purpose hot-plate latencies were also elevated,
but were not statistically
significant,
following
methysergide
given intrathecally
after
doses of 3, 5 and 7.5 mg/kg of morphine using 34 rats. Intrathecal injection of the noradrenergic antagonist phentolamine attenuated the analgesia induced by 2.0 mg/kg of morphine. However, this attenuation was apparent only when analgesia attenuation was surmountable,
was assessed using the hot-plate test. Furthermore, this since phentolamine was unable to antagonize the more
potent analgesia produced by 7.5 mg/kg of morphine. This observation is consistent with our previous report11 which demonstrated that depletion of spinal cord norepinephrine content produced a modest reduction in the capacity of morphine to induce analgesia using the hot-plate, but not the tail-flick test. It is possible that the reduction in morphine analgesia produced by intrathecal phentolamine is a result of the hyperalgesia produced by phentolamine (see Fig. 1). However, this proposal does not seem likely, since analgesia induced by the injection of morphine is not attenuated by the induction of hyperalgesia produced by either the injection of local anesthetics in the nucleus raphe magnuss or depletion of spinal cord serotonin contentl. These findings are consistent with other reports suggesting that noradrenergic neurons participate in mediating opiate analgesiars,is. However, since intrathecally-injected phentolamine was ineffective in altering morphine-induced analgesia using the tail-flick test and only moderately effective using the hot-plate test, it appears that bulbospinal noradrenergic neurons are of minor importance in mediating opiate-induced analgesia. The results of the present study suggest that: (1) tonically active serotonergic and
399 noradrenergic neuronal systems regulate the transmission of nociceptive information at the level of the spinal cord; (2) serotonergic neurons which project to the spinal cord do not participate in the mediation of analgesia induced by systemically administered morphine to an appreciable extent; and (3) noradrenergic neuronal systems which terminate in the spinal cord may mediate a portion of the analgetic action of opiates. We would like to acknowledge the skillful assistance of Ms. Joann Hettasch. This study was supported by Grant NS 12649 to H.K.P.
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