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A spinal site mediates opiate analgesia in frogs
Life Sciences, Vol. 33, pp. 2097-2103 Printed in the U.S.A.
A SPINAL
SITE MEDIATES
Craig W. Stevens
Pergamon Press
OPIATE ANALGESIA
IN FROGS
and Paul D. Pezalla
Department of Biological Sciences U n i v e r s i t y of Illinois at Chicago P.O. Box 4348, Chicago, IL 60680 (Received in final form Aueust 29, 1983) Summary The application of acetic acid to the hind leg of a frog wil] induce a spinally mediated w i p i n g reflex only if the acetic acid concentration is above a certain threshold. By using this reflex as the basis of a test for nociception, we show that morphine sulfate is a potent analgesic in the frog when injected into the lumbar area of the spinal cord. Significant analgesia is induced within 5 min after injection of as little as 0.0316 wg of morphine sulfate. Low doses of morphine sulfate (O.O316 or O.1 ~g) induce analgesia which dissipates within 1 h while for higher doses (O.316,1.O or 3.16 ug) the a n a l g e s i a persists for at least 3 h. The analgesic effect of O.316 Ug of morphine sulfate is completely blocked by naloxone HC1 at either O.158 or O.316 ~g. Animals receiving naloxone alone (O.316 ug) appear to be slightly h y p e r a l g e s i c compared to saline injected controls but this effect is not significant. There is a wealth of information d o c u m e n t i n g the actions of morphine and other opiate drugs at the behavioral and cellular levels. Among these investigations, studies of a n a l g e s i a induced by opiates have yielded the most notable results. Considerable evidence suggests that opiates can mediate analgesia at spinal sites as well as at specific brain loci (I-3). Intrathecal administration of morphine and opioid peptides into the subarachnoid space of the spinal lumbar area produces significant elevation of nociceptive thresholds in a few species of mammals (4-11), including humans (12). It has been shown, that intrathecal morphine depresses ascending nociceptive activity (13) and microelectrophoretically applied morphine generally inhibits n o c i c e p t i v e neurons within the spinal cord (14). In addition to pharmacological studies, a u t o r a d i o g r a p h i c and i m m u n o c y t o c h e m i c a l studies reveal high densities of opiate receptors (15-17) and immunoreactive opioid peptides (18,19) in specific regions of the spinal cord known to process nociceptive information (1,3). A l t h o u g h the amphibian spinal cord has been used extensively in p h a r m a c o l o g i c a l research, few studies have been concerned with the actions of opiates or the functions of endogenous opioids (20). Nistri et al. attempted to relate the a n t i n o c i c e p t i v e action of morphine to its effect on brain and spinal cord acetylcholine levels (21). While these authors did find that 0024-3205/93 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
2098
Spinal Analgesia in Frogs
Vol. 33, No, 2], 1983
morphine slightly elevates a c e t y l c h o l i n e levels, they were unable to detect any effect of morphine on nociceptJve threshold even at doses of 100 mg/kg (21). Zimmerman and Krivoy showed that morphine depresses the monosynaptic ventral root response of the frog isolated spinal cord but concluded that the frog is relatively insensitive to morphine compared to mammals (22). Because endogenous opioid systems appear to be phylogenetically old (23) and because it would not be unreasonable to suppose that they may modulate pain r e s p o n s i v e n e s s in non-mammals, we have reexamined the anslgesic potential of morphine in frogs. In contrast to the findings of Nistri et al. (21), we hsve found that, in frogs, subcutaneous ~orphine induces slight but significant analgesia at 10 mg/kg and pronounced analgesia at 100 mg/kg (24). That this effect is attenuated by naloxone suggests that it is mediated via opiate receptors (24) but the high doses required permit questions c o n c e r n i n g the specificity. We now show that very low doses of morphine produce analgesia when injected directly into the spinal cord and that this effect is completely blocked by naloxone. Materials
and Methods
N o r t h e r n grass frogs, Rana pipiens pipiens (Nasco, Fort Atkinson, WI; mean weight 23g) were housed at 20-22 C in stainless steel a q u a t i c / t e r r e s t r i a l enclosures with running water and a 12 h photoperiod. Frogs were fed live crickets thrice weekly and were acclimated to laboratory conditions for at ]east one week prior to use. On the day before the experiment, frogs were randomly assigned to a treatment group and placed in individual plastic pans (20 X 27 X 15 cm) c o n t a i n i n g water to a depth of 0.5 cm. M o r p h i n e sulfate (kindly supplied by Merck, Sharp and Dohme) and Naloxone HCI (a generous gift from Endo Laboratories) were diluted with physiological saline (110 mM NaC1; 2.3 mM NaHC03; 1.O mM CaC12; pH 7.2) so that the desired dose was delivered in a volume of I ul. For frogs receiving both naloxone and morphine, drugs were delivered in a single volume of I ~I. For radiotracer experiments, tritiated morphine (New England Nuclear, spec. act. 73.9 wCi/mmol) was mixed with non-labelled morphine to yield a final specific activity of 25 ~Ci/3.16 ~g morphine in I ~I. At either 5 or 60 min after intraspinal injection, frogs were quickly frozen in an ethanol/dry ice slurry and spinal segments and brain regions dissected. Each segment was digested in I ml Protosol (New England Nuclear) at 55 C for 24 h and counted for radioactivity. Intraspinal microinjections were made with a Hamilton m i c r o s y r i n g e fitted with a tapered 26g needle (Hamilton point #2). The site of injection was slightly lateral to the neural spine at the articulation between the seventh and eighth vertebrae, the area of the lumbar enlargement of the spinal cord. Penetration of the needle typically produced a mild hind-limb extension or t r e m b l i n g which discontinued after the 2-3 sec delivery of drug. A few frogs showed continued hind-limb extension or trembling after withdrawal of the needle and were considered neurologically damaged and, thus, were not used for the remainder of the experiment. The acetic acid test for a s s e s s i n g
analgesia
in
frogs
has
Vol. 33, No. 21,
1983
Spinal Analgesia in Frogs
2099
been fully described e l s e w h e r e (24). Briefly, serial d ~ l u t i o n s were made to yield ten concentrations of acetic acid equally spaced on a l o g a r i t h m i c scale. The s o l u t i o m ~ were coded, in order of i n c r e a s i n y c o n c e n t r a t i o n s , from I to 10. Code numbers are related to the molar c o n c e n t r a t i o n (M) of acetle acid ~ c c o r d ! n K to the e x p r e s s i o n log M : O.1716 (code number) - 0.5849. NociceFtive testinF was done by pl~cing, with ~ Pasteur pipette, a drop of acetic acid on the dorsa] surface of the frogs thigh. Testing beg~n with the lowest c o n c e n t r a t i o n nnd proceeded with i n c r e a s L n g c~neentrat, ions until t h e n o c i c e p t i v e threshold (UT) w~s reached. The ;:r was defined as the lowest c o n c e n t r a t i o n of acetic ~cid which causes the frog to v l g o r o u s ] y wife the treated leg with either hind limb. To prevent tissue damage, the acetic acid was rinsed off the water after 4 see if the animal failed to respond or immedi~te!y if the anima] resoonded. In ~ few eagles, ~nimsls failed to respond to the highest c o n c e n t r a t i o n of acetic acid nnd the a s s u m F t l o n was made that the animal would respond] to the next highest concentration. Thus, anina~a not resoondinp to concentration 10 were assigned a NT of 1 1 . Dat<~ :~re J i s F ] ~ y e d as either c?::~nFe in NT ( &}iT : p o s t - i n j e c t l o n NT minus ?re-injection NT) or total change in NT ( /AUT for each nnim~]). The
A
II
5 mln
6 0 rain
.
/ q
/
l ÷ ---~K 0
I 00316
I 0.1
l 0.316
i 1.0
I 3.16
i \\ ! 0 OJD316
Morphine (l~g)
J 0.1
! 0.316
I 1.0
L 3.16
Morphlne(IJg)
~IO.
I
D o s e - r e s p o n s e curves obtained 5 min (A) and 60 min (B) after intraspinal i n j e c t i o n of m o r p h i n e sulfate. Each point r e p r e s e n t s the mean ± s.e.m, change in NT from the basal level for a group of IO animals. A s t e r i s k s denote s i g n i f i c a n t l y d i f f e r e n t from saline injected controls at P
2100
Spinal Anal~esia in Frogs
Vol. 33, No. 2], 1983
s i g n i f i c a n c e of treatment effects were assessed using gruskalWallis AN@VA for unrelated samples and, if ~ignific~nt treatment effects were obtained, additional c o m p a r i s o n s were made using the R a n d o m i z a t i o n test (25). Results The dose-response curves o%tained 5 and 60 min after intraspina! ~njection of morphine sulfate are shown in Fly. I while the time course and effect of naloxone are shown in Fig. 2. Kruskal-W~llis A?:OVA revealed n si~n]~tcan~ ~re~tment effe , o_ m o r p h i n e on UT at both 5 min (P
15
12
9
6
5
60 Time after Injection
120 (mi~t)
180
I~g M o ~ 0
0
0.316
0.316
0.316
tag Nal: 0
0.316
0.158
0.316
0
FIG. 2 Intraspinal naloxone (Nal) blockage of the analgesic action of intrasFinal morphine (Nor). (A) Time course of the change in NT (mean ± s.e.m.) after intraspinal injection of 0.316 ug Mot (&, N=8 animals), 0.3~6 UP Mot and 0.158 up Nal (A, N=IO), 0.316 ug Nor and 0.316 Ug Nal ( • , N:8) 0.316 ~g Nal ( B , N=IO), or saline (0, N=6). O v e r l a p p i n g error bars have been omitted. (B) Data from the same experiment plotted as the sum of the change in NT for each animal (mean ± s.e.m.). Asterisks denote significantly different from each of the other groups at P
Vol. 33, No. 21, 1983
Spinal Analgesia in Frogs
2101
changes in NT produced by the three highest doses remained significantly above the controls for the duration of the experiment. No significant changes in NT were ever seen in saline injected animals. The analgesia induced by O.516 Ug of morphine was completely blocked by concurrent intraspina] injection of naloxone at either O . 1 5 8 ug or O.316 ug (Fig. 2). The changes in threshold of animals receiving morphine alone were s i g n i f i c a n t l y greater than those of the controls at 5 min (P
601
A
A. 5 min
i
1o
°6°I M
i ®4 rv 20
0
B. 60 min
~
9 8 7 6 5 4 3 2 ~' MO OL DI CH OF SpinalCord Brain FIG. 3 Distribution of intraspinally injected tritiated morphine at 5 min (A) and 60 min (B) after injection. Data from three frogs at each time are plotted. The spinal cord was cut into nine segments while the brain was grossly divided into five regions; m e d u l l a o b l o n g a t a and cerebellum (MO), optic lobes an& tectum (OL), diencephalon (DI), cerebral hemispheres (CH), and olfactory lobes and tracts (OF).
2102
Spinal Analgesia in Frogs
Vo]. 33, No. 21, 1983
labeled morphine away from the spinal lumbar area was detected. The counts recovered from the various regions of the brain never exceeded 1% of the total recovered radioactivity. Furthermore, no rostral-caudal gradient or other pattern in the d i s t r i b u t i o n of label was noted. Discussion We have previously shown that high doses of systemically administered morphine are required to induce a n a ] g e s i a in frogs (24). Possible reasons for the low potency of systemic morphine include: (a) limited access to the appropriate central nervous system sites, (b) low affinity for the opiate receptor which m e d i a t e s analgesia, (c) low efficacy of morphine in frogs, and (d) lack of an important role of opioid systems in m o d u l a t i n g pain responsiveness of frogs. Our finding that as little as 0.0316 ~g of m o r p h i n e induces significant a n a l g e s i a within 5 mJn indicates that limited access is the dominant, if not sole, reason. A l t h o u g h direct comparisons of our results with published studies on mammals are not possible because of m e t h o d o l o g i c a l differences, we have found significant a n a l g e s i a at doses far lower than the e f f e c t i v e doses of intrathecal morphine in rats. A series of studies by Yaksh and coworkers have shown that the dose p r o d u c i n g half-maximal a n a l g e s i a of morphine administered i n t r a t h e c a l l y in rats was 4-5 wg (7,8,11) whereas we find maximal analgesia at I-3 ~g. However, it is possible that direct intraspinal injection gives a higher c o n c e n t r a t i o n of morphine in the area of spinal opiate receptors m e d i a t i n g a n a l g e s i a or that the nociceptive tests employed have differing sensitivities to morphine. It is unlikely that the analgesic action of intraspina] m o r p h i n e was due to diffusion or rostral transport of the drug to supraspinal sites as we have shown that there is little rostral m o v e m e n t of tritiated morphine away from the spinal lumbar area (Fig. 3). Similar localization of labeled morphine was shown after intrathecal delivery in the rat (8). The present data clearly show a specific a n a l g e s i c action of morphine on spinal opiate receptors because the effect was dosedependent, reversible, and completely blocked by low doses of naloxone. When considered with previous work d o c u m e n t i n g the presence of opioid peptides (26,27) and opiate receptors (28-30) in amphibians, the results reported here suggest an early evolution of endogenous opioid pain modulating systems in vertebrates. Acknowledgements We thank Dr. C h r i s t o p h e r Comer for comments on the manuscript and Marjorie Renfroe for manuscript preparation. Support was provided by the R e s e a r c h Board of the U n i v e r s i t y of Illinois at Chicago. References I. 2. 3.
H.L. FIELDS and A.I. BASBAUM, Ann. Rev. Physiol. 40 217-248 ( 1 978). L.R. WATKINS and D.J. MAYER, Science 216 1185-1792 (1982). T.L. YAKSH and D.L. HAMMOND, Pain I_3_3]-X'~5 (1982).
Vol. 33, No. 21, 1983
4.
A.H.
TANG
and
Spinal Ana]£esia in Frogs
N.J.
SCHOENF£f,D,
Eur.
J.
2103
Ph~rmacol.
52 2 1 5 - 2 2 3
( 1 978). 5. 6. 7. 8. 9. 1 '^', .
T.L. YAKSH, Brain Res. 153 2 0 5 - 2 1 0 (Iq78). [ f~ T.L. YAKSH, R.~. p A. FREDERICF, SO.~;, ,,...° P. !:]ANJ ~n4 m,.A. RUI)Y, B r a i n Res. 1.18 5 1 6 - 5 2 0 (1978). ¢.L. YAKSH snd J.L. HE):RY, Can. J. Physiol. P h q r m a c o ] . 56 7 5 4 - 7 5 9 (I 978). T..T,. YAKS}! and T.A. RUZY, J. P h ~ r m n c o l . ~xp. ~h r q 202 4 ! I - 4 2 8 (1977~. C.J. WCOLF, Brr:in Re~. 189 5 9 3 - 5 9 7 (1980). Pl
J . D.
T,EVI.L'I<,
S.R.
13. 14. 15.
:[.C .
G@RDO:: a n ( ]
r
~! . .T, . . . . . .F. I ~ ' T P q ,
•
Brain
Res 236 85-91 (10~2"I A..t. TU[,'J and ~.L. YAK.~!!, nrqin Re,. 247 75-i~~ (1082). ". , _~vAr:,~, T. J-E_., n. YAFIAYA, . . fl . LI,qC . and ~> 3 U I L L E F I N , Lancer, I "~2-124 /lO90'. T . DOI a n d I . JURf,A, 9 r ~ . i n R e s . 234 Z 9 " ~ - 4 0 7 ( 1 9 - ~ 2 ) . 3. SELCH:-,I{ and !{.W. R Y A L L , £ r q i n R e s . 145 7 0 3 - 3 1 4 !1978). C. LAI.ICTTE, C. "~ F?:RT an,J t . H . S::YEF:E, 2 r a i n Re~. ~12 4 0 7 - 4 1 2 •
14 . I °=
LA:[[':,
p
.
.
(I 9,7c.',.
. . . . . . . ~,~r.+. and r:.J. kJ;IAR. ~rsin r,,~ 124 53-67 (1077) 17 [4. ," I N ~...O. . .v ' P q ~ HU:~T ~nd J R.W. G~...~.V:., " ~. . . . Brnin R r s . 241 197-206 (1982). 18. z.J. .31PSCI,:, J . M . PCLAi<, ? . R . T.LOCPI and P . D . WALL, ,1. Ccmp. N e u r o l . 201 6 5 - 7 9 (108,' ~,. 19. P.M. :'WE£'21;AP', J . ! { . "',,£AL,,,m ,'.T~. EARKER an,4 A. ~'.,OLD~r-":,, Proc. tistl. Acad. Sol. U.t.A. 79 6 7 4 2 - 6 7 4 6 (1982). 20. Y. KU[~C, Pro~. ~Jeurobio]. :~ I-7~--(1978). Pl A. ~llq~RI G. PEPEU, E. CAr,IMELL[ L. ~pTNA and A.[.I. ~:" P:-LLI~, Brain Res. 80 1 9 9 - 2 0 9 (197~). •....."-.R,,AN.~ Yed 146 22. L. .... ,~""~, . . . : a n d W. Z R I V n v Proc L',oc .:':xn • ~io] 575-529 (1974). 23 . C. Re~ . 75 356-361 . ~ . PERT, . . D. . A P O. S H I.A N , . ~ }~. ~ , Iv. D ~~'f4 _, 9r~tn •
±.F
t
, . . . .
.
/ 1Q7,4 x
24. 25.
26. 27. 28. ~,.°q
30.
F.D. PEZALLA, Brain Res. (~n press). ...... ~:onparametric_ S t a t i s t [ c s for the B e h a v i o r ~ a ] ° ° IE~E~, ~ " .qctences, Y c C r e w - H i l l , .few Ycrk, T]-95--~,. P.D. PEZALLA, N.G. ,qEIDAF, S. P~ENJANNFT, P. GRIllE, t,. LIS and M ~.{E.._:EN Life Sci 23 2 2 8 1 - 2 2 q 2 (1Q'7~) R.I. CONE and A. O O L D S T E ! N , Proc f,.,tl. Acmd. P,ci. U.fi A. 79 3345-3349 (1982'). Y. AUDIG!k'R, A.[,I. D U P R A T ~nd J. CECS, Comp. Biocher. Physiol. 67 C 1 9 1 - 1 9 3 (1980) U . T . RUEG3, S. CUENOD, J . t { . MILL.El{, =.. G I O A N N I [ [ I , R.D. HOWELLS a n d E . J . SIMON, Proc. Natl. Acad. 2ci. U.S.A. 78 4635-4638 (198~). E.J. SIMON, J.M. IilLLER, J. GROTH, Y. ITZHA~', M. J. H O L L A N D and S.C. BECK, Life Sei. 3 1 136"7-13"70 (1982).
A SPINAL
SITE MEDIATES
Craig W. Stevens
Pergamon Press
OPIATE ANALGESIA
IN FROGS
and Paul D. Pezalla
Department of Biological Sciences U n i v e r s i t y of Illinois at Chicago P.O. Box 4348, Chicago, IL 60680 (Received in final form Aueust 29, 1983) Summary The application of acetic acid to the hind leg of a frog wil] induce a spinally mediated w i p i n g reflex only if the acetic acid concentration is above a certain threshold. By using this reflex as the basis of a test for nociception, we show that morphine sulfate is a potent analgesic in the frog when injected into the lumbar area of the spinal cord. Significant analgesia is induced within 5 min after injection of as little as 0.0316 wg of morphine sulfate. Low doses of morphine sulfate (O.O316 or O.1 ~g) induce analgesia which dissipates within 1 h while for higher doses (O.316,1.O or 3.16 ug) the a n a l g e s i a persists for at least 3 h. The analgesic effect of O.316 Ug of morphine sulfate is completely blocked by naloxone HC1 at either O.158 or O.316 ~g. Animals receiving naloxone alone (O.316 ug) appear to be slightly h y p e r a l g e s i c compared to saline injected controls but this effect is not significant. There is a wealth of information d o c u m e n t i n g the actions of morphine and other opiate drugs at the behavioral and cellular levels. Among these investigations, studies of a n a l g e s i a induced by opiates have yielded the most notable results. Considerable evidence suggests that opiates can mediate analgesia at spinal sites as well as at specific brain loci (I-3). Intrathecal administration of morphine and opioid peptides into the subarachnoid space of the spinal lumbar area produces significant elevation of nociceptive thresholds in a few species of mammals (4-11), including humans (12). It has been shown, that intrathecal morphine depresses ascending nociceptive activity (13) and microelectrophoretically applied morphine generally inhibits n o c i c e p t i v e neurons within the spinal cord (14). In addition to pharmacological studies, a u t o r a d i o g r a p h i c and i m m u n o c y t o c h e m i c a l studies reveal high densities of opiate receptors (15-17) and immunoreactive opioid peptides (18,19) in specific regions of the spinal cord known to process nociceptive information (1,3). A l t h o u g h the amphibian spinal cord has been used extensively in p h a r m a c o l o g i c a l research, few studies have been concerned with the actions of opiates or the functions of endogenous opioids (20). Nistri et al. attempted to relate the a n t i n o c i c e p t i v e action of morphine to its effect on brain and spinal cord acetylcholine levels (21). While these authors did find that 0024-3205/93 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
2098
Spinal Analgesia in Frogs
Vol. 33, No, 2], 1983
morphine slightly elevates a c e t y l c h o l i n e levels, they were unable to detect any effect of morphine on nociceptJve threshold even at doses of 100 mg/kg (21). Zimmerman and Krivoy showed that morphine depresses the monosynaptic ventral root response of the frog isolated spinal cord but concluded that the frog is relatively insensitive to morphine compared to mammals (22). Because endogenous opioid systems appear to be phylogenetically old (23) and because it would not be unreasonable to suppose that they may modulate pain r e s p o n s i v e n e s s in non-mammals, we have reexamined the anslgesic potential of morphine in frogs. In contrast to the findings of Nistri et al. (21), we hsve found that, in frogs, subcutaneous ~orphine induces slight but significant analgesia at 10 mg/kg and pronounced analgesia at 100 mg/kg (24). That this effect is attenuated by naloxone suggests that it is mediated via opiate receptors (24) but the high doses required permit questions c o n c e r n i n g the specificity. We now show that very low doses of morphine produce analgesia when injected directly into the spinal cord and that this effect is completely blocked by naloxone. Materials
and Methods
N o r t h e r n grass frogs, Rana pipiens pipiens (Nasco, Fort Atkinson, WI; mean weight 23g) were housed at 20-22 C in stainless steel a q u a t i c / t e r r e s t r i a l enclosures with running water and a 12 h photoperiod. Frogs were fed live crickets thrice weekly and were acclimated to laboratory conditions for at ]east one week prior to use. On the day before the experiment, frogs were randomly assigned to a treatment group and placed in individual plastic pans (20 X 27 X 15 cm) c o n t a i n i n g water to a depth of 0.5 cm. M o r p h i n e sulfate (kindly supplied by Merck, Sharp and Dohme) and Naloxone HCI (a generous gift from Endo Laboratories) were diluted with physiological saline (110 mM NaC1; 2.3 mM NaHC03; 1.O mM CaC12; pH 7.2) so that the desired dose was delivered in a volume of I ul. For frogs receiving both naloxone and morphine, drugs were delivered in a single volume of I ~I. For radiotracer experiments, tritiated morphine (New England Nuclear, spec. act. 73.9 wCi/mmol) was mixed with non-labelled morphine to yield a final specific activity of 25 ~Ci/3.16 ~g morphine in I ~I. At either 5 or 60 min after intraspinal injection, frogs were quickly frozen in an ethanol/dry ice slurry and spinal segments and brain regions dissected. Each segment was digested in I ml Protosol (New England Nuclear) at 55 C for 24 h and counted for radioactivity. Intraspinal microinjections were made with a Hamilton m i c r o s y r i n g e fitted with a tapered 26g needle (Hamilton point #2). The site of injection was slightly lateral to the neural spine at the articulation between the seventh and eighth vertebrae, the area of the lumbar enlargement of the spinal cord. Penetration of the needle typically produced a mild hind-limb extension or t r e m b l i n g which discontinued after the 2-3 sec delivery of drug. A few frogs showed continued hind-limb extension or trembling after withdrawal of the needle and were considered neurologically damaged and, thus, were not used for the remainder of the experiment. The acetic acid test for a s s e s s i n g
analgesia
in
frogs
has
Vol. 33, No. 21,
1983
Spinal Analgesia in Frogs
2099
been fully described e l s e w h e r e (24). Briefly, serial d ~ l u t i o n s were made to yield ten concentrations of acetic acid equally spaced on a l o g a r i t h m i c scale. The s o l u t i o m ~ were coded, in order of i n c r e a s i n y c o n c e n t r a t i o n s , from I to 10. Code numbers are related to the molar c o n c e n t r a t i o n (M) of acetle acid ~ c c o r d ! n K to the e x p r e s s i o n log M : O.1716 (code number) - 0.5849. NociceFtive testinF was done by pl~cing, with ~ Pasteur pipette, a drop of acetic acid on the dorsa] surface of the frogs thigh. Testing beg~n with the lowest c o n c e n t r a t i o n nnd proceeded with i n c r e a s L n g c~neentrat, ions until t h e n o c i c e p t i v e threshold (UT) w~s reached. The ;:r was defined as the lowest c o n c e n t r a t i o n of acetic ~cid which causes the frog to v l g o r o u s ] y wife the treated leg with either hind limb. To prevent tissue damage, the acetic acid was rinsed off the water after 4 see if the animal failed to respond or immedi~te!y if the anima] resoonded. In ~ few eagles, ~nimsls failed to respond to the highest c o n c e n t r a t i o n of acetic acid nnd the a s s u m F t l o n was made that the animal would respond] to the next highest concentration. Thus, anina~a not resoondinp to concentration 10 were assigned a NT of 1 1 . Dat<~ :~re J i s F ] ~ y e d as either c?::~nFe in NT ( &}iT : p o s t - i n j e c t l o n NT minus ?re-injection NT) or total change in NT ( /AUT for each nnim~]). The
A
II
5 mln
6 0 rain
.
/ q
/
l ÷ ---~K 0
I 00316
I 0.1
l 0.316
i 1.0
I 3.16
i \\ ! 0 OJD316
Morphine (l~g)
J 0.1
! 0.316
I 1.0
L 3.16
Morphlne(IJg)
~IO.
I
D o s e - r e s p o n s e curves obtained 5 min (A) and 60 min (B) after intraspinal i n j e c t i o n of m o r p h i n e sulfate. Each point r e p r e s e n t s the mean ± s.e.m, change in NT from the basal level for a group of IO animals. A s t e r i s k s denote s i g n i f i c a n t l y d i f f e r e n t from saline injected controls at P
2100
Spinal Anal~esia in Frogs
Vol. 33, No. 2], 1983
s i g n i f i c a n c e of treatment effects were assessed using gruskalWallis AN@VA for unrelated samples and, if ~ignific~nt treatment effects were obtained, additional c o m p a r i s o n s were made using the R a n d o m i z a t i o n test (25). Results The dose-response curves o%tained 5 and 60 min after intraspina! ~njection of morphine sulfate are shown in Fly. I while the time course and effect of naloxone are shown in Fig. 2. Kruskal-W~llis A?:OVA revealed n si~n]~tcan~ ~re~tment effe , o_ m o r p h i n e on UT at both 5 min (P
15
12
9
6
5
60 Time after Injection
120 (mi~t)
180
I~g M o ~ 0
0
0.316
0.316
0.316
tag Nal: 0
0.316
0.158
0.316
0
FIG. 2 Intraspinal naloxone (Nal) blockage of the analgesic action of intrasFinal morphine (Nor). (A) Time course of the change in NT (mean ± s.e.m.) after intraspinal injection of 0.316 ug Mot (&, N=8 animals), 0.3~6 UP Mot and 0.158 up Nal (A, N=IO), 0.316 ug Nor and 0.316 Ug Nal ( • , N:8) 0.316 ~g Nal ( B , N=IO), or saline (0, N=6). O v e r l a p p i n g error bars have been omitted. (B) Data from the same experiment plotted as the sum of the change in NT for each animal (mean ± s.e.m.). Asterisks denote significantly different from each of the other groups at P
Vol. 33, No. 21, 1983
Spinal Analgesia in Frogs
2101
changes in NT produced by the three highest doses remained significantly above the controls for the duration of the experiment. No significant changes in NT were ever seen in saline injected animals. The analgesia induced by O.516 Ug of morphine was completely blocked by concurrent intraspina] injection of naloxone at either O . 1 5 8 ug or O.316 ug (Fig. 2). The changes in threshold of animals receiving morphine alone were s i g n i f i c a n t l y greater than those of the controls at 5 min (P
601
A
A. 5 min
i
1o
°6°I M
i ®4 rv 20
0
B. 60 min
~
9 8 7 6 5 4 3 2 ~' MO OL DI CH OF SpinalCord Brain FIG. 3 Distribution of intraspinally injected tritiated morphine at 5 min (A) and 60 min (B) after injection. Data from three frogs at each time are plotted. The spinal cord was cut into nine segments while the brain was grossly divided into five regions; m e d u l l a o b l o n g a t a and cerebellum (MO), optic lobes an& tectum (OL), diencephalon (DI), cerebral hemispheres (CH), and olfactory lobes and tracts (OF).
2102
Spinal Analgesia in Frogs
Vo]. 33, No. 21, 1983
labeled morphine away from the spinal lumbar area was detected. The counts recovered from the various regions of the brain never exceeded 1% of the total recovered radioactivity. Furthermore, no rostral-caudal gradient or other pattern in the d i s t r i b u t i o n of label was noted. Discussion We have previously shown that high doses of systemically administered morphine are required to induce a n a ] g e s i a in frogs (24). Possible reasons for the low potency of systemic morphine include: (a) limited access to the appropriate central nervous system sites, (b) low affinity for the opiate receptor which m e d i a t e s analgesia, (c) low efficacy of morphine in frogs, and (d) lack of an important role of opioid systems in m o d u l a t i n g pain responsiveness of frogs. Our finding that as little as 0.0316 ~g of m o r p h i n e induces significant a n a l g e s i a within 5 mJn indicates that limited access is the dominant, if not sole, reason. A l t h o u g h direct comparisons of our results with published studies on mammals are not possible because of m e t h o d o l o g i c a l differences, we have found significant a n a l g e s i a at doses far lower than the e f f e c t i v e doses of intrathecal morphine in rats. A series of studies by Yaksh and coworkers have shown that the dose p r o d u c i n g half-maximal a n a l g e s i a of morphine administered i n t r a t h e c a l l y in rats was 4-5 wg (7,8,11) whereas we find maximal analgesia at I-3 ~g. However, it is possible that direct intraspinal injection gives a higher c o n c e n t r a t i o n of morphine in the area of spinal opiate receptors m e d i a t i n g a n a l g e s i a or that the nociceptive tests employed have differing sensitivities to morphine. It is unlikely that the analgesic action of intraspina] m o r p h i n e was due to diffusion or rostral transport of the drug to supraspinal sites as we have shown that there is little rostral m o v e m e n t of tritiated morphine away from the spinal lumbar area (Fig. 3). Similar localization of labeled morphine was shown after intrathecal delivery in the rat (8). The present data clearly show a specific a n a l g e s i c action of morphine on spinal opiate receptors because the effect was dosedependent, reversible, and completely blocked by low doses of naloxone. When considered with previous work d o c u m e n t i n g the presence of opioid peptides (26,27) and opiate receptors (28-30) in amphibians, the results reported here suggest an early evolution of endogenous opioid pain modulating systems in vertebrates. Acknowledgements We thank Dr. C h r i s t o p h e r Comer for comments on the manuscript and Marjorie Renfroe for manuscript preparation. Support was provided by the R e s e a r c h Board of the U n i v e r s i t y of Illinois at Chicago. References I. 2. 3.
H.L. FIELDS and A.I. BASBAUM, Ann. Rev. Physiol. 40 217-248 ( 1 978). L.R. WATKINS and D.J. MAYER, Science 216 1185-1792 (1982). T.L. YAKSH and D.L. HAMMOND, Pain I_3_3]-X'~5 (1982).
Vol. 33, No. 21, 1983
4.
A.H.
TANG
and
Spinal Ana]£esia in Frogs
N.J.
SCHOENF£f,D,
Eur.
J.
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Ph~rmacol.
52 2 1 5 - 2 2 3
( 1 978). 5. 6. 7. 8. 9. 1 '^', .
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S.R.
13. 14. 15.
:[.C .
G@RDO:: a n ( ]
r
~! . .T, . . . . . .F. I ~ ' T P q ,
•
Brain
Res 236 85-91 (10~2"I A..t. TU[,'J and ~.L. YAK.~!!, nrqin Re,. 247 75-i~~ (1082). ". , _~vAr:,~, T. J-E_., n. YAFIAYA, . . fl . LI,qC . and ~> 3 U I L L E F I N , Lancer, I "~2-124 /lO90'. T . DOI a n d I . JURf,A, 9 r ~ . i n R e s . 234 Z 9 " ~ - 4 0 7 ( 1 9 - ~ 2 ) . 3. SELCH:-,I{ and !{.W. R Y A L L , £ r q i n R e s . 145 7 0 3 - 3 1 4 !1978). C. LAI.ICTTE, C. "~ F?:RT an,J t . H . S::YEF:E, 2 r a i n Re~. ~12 4 0 7 - 4 1 2 •
14 . I °=
LA:[[':,
p
.
.
(I 9,7c.',.
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, . . . .
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/ 1Q7,4 x
24. 25.
26. 27. 28. ~,.°q
30.
F.D. PEZALLA, Brain Res. (~n press). ...... ~:onparametric_ S t a t i s t [ c s for the B e h a v i o r ~ a ] ° ° IE~E~, ~ " .qctences, Y c C r e w - H i l l , .few Ycrk, T]-95--~,. P.D. PEZALLA, N.G. ,qEIDAF, S. P~ENJANNFT, P. GRIllE, t,. LIS and M ~.{E.._:EN Life Sci 23 2 2 8 1 - 2 2 q 2 (1Q'7~) R.I. CONE and A. O O L D S T E ! N , Proc f,.,tl. Acmd. P,ci. U.fi A. 79 3345-3349 (1982'). Y. AUDIG!k'R, A.[,I. D U P R A T ~nd J. CECS, Comp. Biocher. Physiol. 67 C 1 9 1 - 1 9 3 (1980) U . T . RUEG3, S. CUENOD, J . t { . MILL.El{, =.. G I O A N N I [ [ I , R.D. HOWELLS a n d E . J . SIMON, Proc. Natl. Acad. 2ci. U.S.A. 78 4635-4638 (198~). E.J. SIMON, J.M. IilLLER, J. GROTH, Y. ITZHA~', M. J. H O L L A N D and S.C. BECK, Life Sei. 3 1 136"7-13"70 (1982).