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Analgesic brain stimulation in the cat: Effect of intraventricular serotonin, norepinephrine, and dopamine
EXPERIMENTAL
NEUROLOGY
57, 1059-1066 (1977)
RESEARCH Analgesic Brain lntraventricular
DAVID
NOTE
Stimulation in the Cat: Effect Serotonin, Norepinephrine, and Dopamine
Duu~~sso~
AFXI RONALD
~ELZACK
of
’
It is now firmly established that electrical stimulation of structures in or near the mesencephalic central gray matter produces analgesia in discrete parts of the body (2, 6, 15, 16, 18-20, 22, 23, 25). Furthermore, microinjections of morphine into the ventricles or into periventricular regions produce analgesia, whereas similar injections into a variety of other sites are ineffective (12, 13, 24, 30, 36), which suggests that the action of opiate analgesic drugs is similar to that of electrical stimulation. A synergistic effect has been demonstrated between subanalgesic doses of morphine and stimulation of the dorsal raphe nucleus in rats (29)) and Mayer and Hayes (17) showed cross-tolerance to morphine and analgesic brain stimulation. Recently Akil cf al. (4) reported antagonism to analgesic brain stimulation by naloxone, a potent narcotic antagonist. The major effects of morphine have been attributed to its interaction with aminergic pathways in the brain (14, 39). In the rat, depletion of monoaminesby tetrabenazine, reserpine, and other drugs, or specific inhibition of serotonin synthesis by p-chlorphenylalanine, antagonizes both stimulation-produced analgesia (2, 3, 16) and morphine analgesia (7, 8, 35, 39.) The dopamine receptor stimulator, apomorphine, potentiates stimulation-produced analgesia, whereas blockade of dopamine receptors by Abbreviations : DOPA-dihydroxyphenylalanine. 1 Supported by Grant A7891 from the National Research Council of Canada. Dr. Dubuisson was supported by a student fellowship from Quebec Medical Research Council. The authors thank George Nieman and Warren Soper for technical assistance. Address reprint requests to Dr. Ronald Melzack, Department of Psychology, McGill University, 1205 McGregor Avenue, Montreal, PQ, Canada H3A 1Bl. 1059 Copyright Q 1977 by AcademicPress,Inc. All rights of reproduction in any form reserved.
ISSN
0014-4586
1060
DUXUISSON
AND
MELZACK
pimozide causes its reduction (2). Akil and Mayer (3) speculated that the analgesia produced by morphine and electrical stimulation in the ventral portion of the periaqueductal gray results from activation of a neural substrate which contains at least one serotonergic synapse. Evidence in support of this is provided by studies involving lesions of the medial forebrain bundle (11, 40)) administration of biogenic amines or their precursors (2, 8, 34), and measurements of forebrain serotonin content after stimulation or destruction of the midbrain raphe nuclei (1, 27, 28, 31, 32), from which a major complement of serotonergic axons is known to originate (5, 10, 37). Furthermore, Oliveras et nl. (23) noted profound analgesia produced by stimulation of a serotonin-rich region of the caudal medulla. Although the serotonergic link implicated by Akil and Mayer could ascend to the forebrain or descend to the cord from lower brain stem structures, it has been theorized (2, 3, 15, 16, 26) that morphine and analgesic brain stimulation have an ultimate common site of action in blocking nociceptive input through the dorsal horn system, a concept based on the gate control theory of Melzack and Wall (21). Sparkes and Spencer (34) measured the strength of morphine analgesia in rats after intraventricular injections of serotonin, norepinephrine, dopamine, and DOPA (dihydroxyphenylalanine) . They found that serotonin potentiated morphine analgesia, and norepinephrine, DOPA, and dopamine reduced the effectiveness of morphine. However, Calcutt et al. (S) reported that intraventricular dopamine facilitated morphine analgesia in the rat. No previous study of stimulation-produced analgesia has utilized direct intraventricular injections of biogenic amines. The present study examines the effect on stimulation-produced analgesia of intraventricular serotonin, norepinephrine, and dopamine. The subjects were 25 female cats weighing 2.4 to 3.8 kg. Under ketamine anesthesia, insulated bipolar stainless-steel electrodes were implanted stereotactically toward a point in the nucleus raphe dorsalis which is known (22) to evoke consistent analgesia. Target coordinates, based on the atlas of Snider and Niemer (33), were P-0.5, L-0.0, V-0.5. In four animals, stainless-steel cannulae were also inserted stereotactically to a point in the right lateral ventricle, 13.0 mm anterior to the vertical interaural plane. Stimulating currents consisted of biphasic rectangular p&es of 20 to 330 pA at a frequency of 30 Hz and pulse duration of 2 ms. Animals were pretested at least 24 h before the treatment sessionto determine the current levels which would produce intense analgesia to pinch with a hemostat, with a minimum of extraneous behavior such as tremor, circling, or escape. During observation, the animals were free to move about in a 75 X 75 X go-cm chamber with a Plexiglas door. A lightweight harness and leash,
ntlnclictl ;11~(1~~c 10 a rotatiug ass~i~~hl~-. allowetl tlir aniiiials to sit, walk ahut, aud explore the observation cliaiiil)er, hut prevented then1 from lyiiig down. Current was delivered through a conmutator and flexible cord. The analgesic effect of stimulation alone was observed in six animals. On the treatment day, each animal was allowed 5 min to become accustomed to the observation cliainl~er before receiviug 0.1 ml S>h formalin solution, froni a lxessurized tramcutaneous inj&or, iii the anteriormost portion of the main pad of the right front paw. Stimulation then began, and a continuous record uf pain intensity- ratings was kept on a polygraph with the aid of a four-position switch. Pain intensity was evaluated as follows : 3-licking or biting paw; 2-holding paw off floor; l-paw resting lightly on floor, not bearing weight; O-paw pressed flat on floor, dorsiflexed, with no observable difference from opposite paw. Mean paiu intensities for 3-min intervals were calculated from the polygraph record. A group of nine animals served as controls, receiving only the formalin injection. A third group, consisting of six animals, underwent a similar testing procedure except that 5 mill before the formalin injection, morphine sulfate (0.8 lug/kg) was administered intraperitoneally, and the stimulating current remained off. Observation periods lasted 30 min. A final group of four animals received a series of four treatments spaced 2 weeks apart and assigned in random order. Treatments consisted of observation periods lasting 30 niin after injection of formalin into a forel)aw ; during alternate sessions, the injection was made into the left paw rather than the right. Fifteen minutes prior to formalin injection, one of the following substances was administered into the lateral ventricle : (i) serotoniii-creatiiiine sulfate, 200 pg ; (ii) norepinephrine bitartrate, SO pg ; (iii) dopanke hydrochloride, 60 pg ; or (iv) physiological saline solution, as a control procedure. Intraventricular injections of amines in similar concentrations have been shown (9 j to have rapid, potent physiological effects in the cat. Injections were made from disposable syringes through a suitable adaptor. All compounds were dissolved in sterile, pyrogen-free physiologic saline so that the required amount of amine was contained in 0.2 nil solution. This was washed in immediately by a second injection of 0.15 ml saline. Iii the case of controls. a siiigle injection was made of 0.35 ml saline. Brain stimulation was delievered as described above, aud pain ratings were averaged for 3-miii intervals. Each animal was observed under each of the four treatment conditions. At the termination of the experiment, the animals were killed and electrode placements (Fig. 1) were determined by standard histological techniques. The position of the cannula tip within the lateral ventricle was
1062
DUBUISSON
AND
MELZACK
Frc. 1. Electrode placements which produccd analgesia in animals receiving stimulation alone (closed circles) or stimulation plus intraventricular injections (open circles).
verified by gross dissection of the fixated cerebrum prior to sectioning of the brain stem. Figure 2a shows the change in pain intensity during a 30-min period in the control animals, as well as the effects of stimulation and morphine. Both analgesic treatments clearly reduced the average pain rating during the first lo-min period, as animals began to ignore the injected paw and to place weight upon it. Figure 2b shows that dopamine or norepinephrine given intraventricularly reduced the effectivness of analgesic brain stimulation. After administration of either of these amines, the animals did not show typical analgesia, but rather tended to avoid using the injected paw throughout most of the 30-min testing period. In addition, pinch tests at the conclusion of each observation period disclosed that all animals which had received norepinephrine, and all but one of the animals which had received dopamine, showed normal withdrawal of the forepaws despite continuing brain stimulation. The combination of serotonin and brain stimulation produced unusual disturbances of motor tone which interfered with the pain rating system. In the 15-min interval prior to the formalin injection, the animals showed only mild somnolence and motor weakness. They adopted a wide stance and attempted to lie down, but were prevented from doing so by the harness. At the onset of stimulation, however, three of four animals dis-
FIG. 2. Pain intensity ratings after injection of formalin into ,the forepaw averaged for groups of animals. a-Pain rating curves for animals that received morphine or periaqueductal gray stimulation (P.4G) in comparison to controls. b-Pain ratings for animals that received stimulation plus intraventricular norepinephrine (NOR) or dopamine (DOP) versus stimulation plus intraventricular saline (S.AL).
played characteristic bizarre, cataleptiform posturing which persisted throughout the observation period. One animal crouched, motionless, with all four paws on the floor ; another showed rigid extension of both front legs; and a third held the injected paw straight and immobile at a level above its own head. The remaining animal demonstrated rapid and clear-cut analgesia with no abnormality of muscle tone. Thus, three of four animals placed weight on the injected paw throughout most of the observation period. Firm pressure with a hemostat at ‘the end of the observation period produced minimal reaction in all four animals, which suggests that analgesia was incleed present, but confounded by the neuromuscular disturbance.
1064
DUl3UISSON
AND
MELZACK
The results indicate that intraventricular norepiucpllrinc and dopamine arc autagouistic to analgesic braiu stiniulation in the cat. The findings concerning serotonin suggest a combination of analgesia and disruption of motor tone. This picture resembles the work of Sparkes and Spencer (34) on morphine analgesia. The results of intraventricular dopamine conflict with those of Akil and Liebeskind (2), who observed in the rat a potentiation of stimulation-produced analgesia by pharmacologic agents which exert a predominant central dopaminergic influence. The mechanism of action of intraventricular dopamine may be different from that of peripherally administered L-DOPA after depletion of brain amines or from that of apomorphine (2). Although the effects of biogenic amines in the present study are consistent with the concept of a common neural substrate for morphine and central gray stimulation. no single neurotransmitter can be equated with the neural mechanisms underlying narcotic analgesia. The behavior of individual animals suggests that a continua1 balance of aminergic activity may be altered in several ways either to permit or to disrupt analgesia. In conclusion, the results show that no single aminergic neurotransmitter can be equated with the neural mechanism underlying stimulation-produced analgesia. Rather, there appears to be a balance of serotonergic and catecholaminergic influences. REFERENCES 1. AGHAJANIAN, G. K., J. A. ROSECRANS, AND M. H. SHEARD. 1967. Serotonin: Release in the forebrain by stimulation of midbrain raphe. S&wcc 156: 706-708. 2. AI(IL, H., AND J. C. LIEBESKIND. 1975. Monoaminergic mechanisms of stimulationproduced analgesia. Brain Rcs. 94 : 279-296. 3. AKIL, H., AND D. J. MAYER. 1972. Antagonism of stimulation-produced analgesia by p-CPA, a serotonin synthesis inhibitor. Brain Rcs. 44 : 692-697. 4. AKIL, H., D. J. MAYER, AND J. C. LIEBESKIND. 1976. Reduction of stimulationproduced analgesia by the narcotic antagonist, naloxone. Sciolce 191: 961-962. 5. ANDEN, N. E., A. DAHLSTROM, K. FUXE, K. LARSSON, L. OLSON, AND U. UNGERSTEDT. 1966. Ascending monoamine neurons to the telencephalon and diencephalon. Acta Physiol. Stand. 67 : 313326. 6. BALAGURA, S., AND T. RALPH. 1973. The analgesic effect of electrical stimulation of the diencephalon and mesencephalon. BrailL Res. 60 : 369-379. 7. BAPAT, S. K., AND V. CHANDRA. 1968. Effect of tetrabenazine on morphine analgesia in rats. ImGun J. Plzysiol. Phanrcacol. 12 : 107-109. 8. CALCUTT, C. R., N. S. DOGGETT, AND P. S. J. SPENCER. 1971. Modification of the antinociceptive activity of morphine by centrally administered ouabain and dopamine. Psycltopharntacologia 21 : 11 l-l 17. 9. FELDBERG, W., AND S. L. SHERWOOD. 1954. Injections of drugs into the lateral ventricle of the cat. J. Pltysiol. (Land.) 123 : 148-167. 10. FUXE, K. 1965. Distribution of monoamine nerve terminals in the central nervous system. Acta PAysiol. Stand. 64, [Suppl. 2471 : 30-84.
CATECHOLAI\IINES
Ir;
BRAIN
ANALtiESIA
106.5
11. II.\IIYFY. J. :I., .zx;n C. E. LISTS. 1971. Lesions in the medial forebrain hundlc: Relationship bet\veen pain sensitivity and telencephalic content of scrotonin. .I. Camp. Physiol. PsychoI. 74 : 28-36. 12. HIXZ, .4., K. ALBUS, J. METYS, P. SCHUBERT, AKD H. J. TESCHEJIACHER. 1970. On the central sites for the antinociceptive action of morphine and fentanyl. Nclrropknrrllacol(,1/3’ 9 : 539-551. 13. JACQ!I~ET, Y. I;., ~ZND A. l.AJ’rII:1. 1973. ?*lorphinc action at central nervous system sites in the rat: Analgesia or hyperalgesia depending on site and dose. Scirnrc 182 : 490-491. 14. I.EE, J. R., ASD M. R. FER.X.ESSY. 1970. The relationship between morphine analgesia and the levels of biogenic amines in the mouse brain. El/u. J. Pharfrlaco[. 12: 65-70. 15. LIEBESKIND, J. C., G. G~ILBA~D, J. M. BESSON, AND J. L. OLIVERAS. 1973. Analgesia from electrical stimulation of the periaqueductal grey matter in the cat: Behavioral observations and inhibitory effects on spinal cord interneurons. Bvoirz Rcs. 50 : 441-446. 16. LIEBESKIND, J. C., D. J. MAYER, AND H. AKIL. 1974. Central mechanisms of pain inhibition: Studies of analgesia from focal brain stimulation. Pages 261-268 in J, J. BONICA, Ed., .4d7farlcrs in Nmrolog~l, 7’01. J: Pai~. Raven Press, New York. 17. MAYER, D. J., AND R. HAYES. 1975. Stimulation-produced analgesia: Development of tolerance and cross-tolerance to morphine. Scirrrcc 188 : 941-943. 18. MAYER, D. J., AP;D J. C. LIEBESKKD. 1974. Pain reduction by focal electrical stimulation of the brain: -4n anatomical and behavioral analysis. Brni~ Rrs. 68: 73-93. 19. MAUER, D. J., T. L. WOLFLE, H. AKIL, B. CARDER, AND J. C. LIEBESKIND. 1971. Analgesia from electrical stimulation in the brainstem of the rat. Scict~cc 174: 1351-1354. 20. MELZACK, R., AND D. F. MELIXKOPF. 1974. iZnalgesia produced by brain stimulation : Evidence of a prolonged onset period. Exp. Nrrrrol. 43 : 369-374. 21. MIXZACK, R., AND P. D. WAI.L. 1965. Pain mechanisms: ?, new theory. Scirt~cr 150 : 971-979. 22. OLIVERAS, J. I,., J. M. BESSON, G. GCILBATD, AND J. C. LIEBESKIND. 1974. Behavioral and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat. Exfi. Brairt Rrs. 20 : 32-44. 23. OI.IVERAS, J. L., F. REDJEMI, G. GI:II.BAVD. AxD J. M. BUSOX. 1975. Analgesia induced by electrical stimulation of the inferior centralis nucleus of the raphe in the cat. Pairl 1: 139-146. 24. PERT, A., AND T. YAKSH. 1974. Sites of morphine-induced analgesia in the primate brain: Relation to pain pathways. Bvaitr Rcs. 80: 135-140. 25. REYNOI.DS, D. V. 1969. Surgery in the rat during electrical stimulation induced by focal brain stimulation. S&rcc 164 : 444445. 26. RHODES, D. L., AKD J. C. LIEBESKIND. 1976. The central mode of action of narcotic analgesic drugs. Pages 143-152 in M. E. JARVICK, Ed., Ps~‘clzophar;r~ocology in the Practice of Mcdicirw. Appleton-Century-Crofts, New York. 27. SAMANN, R., AND S. BEREASCONI. 1972. Effects of intraventricularly injected 6-OH dopamine or midbrain raphe lesions on morphine analgesia in rats. Psycl~oplmrruacologia 25 : 175-182. 28. SAMANIN, R., W. GCMULKA, AKD L. VALZELLI. 1970. Reduced effect of morphine in midbrain raphe lesioned rats. Eu~. J. Pllarlrzucol. 10 : 339-343.
1066 29. 30.
31. 3.2. 33. 34.
35. 36. 37. 38. 39.
40.
DUBUISSON
AND
MELZACK
IN, R., AND 1,. \rAr.zEr,r.r. 1971. Increase of morl)hitlc-intlucctl annlgrsin hy stimulation of the nucleus raphe dorsalis. Em-. J. Phwncol. 16 : 298-302. SHARPE, L. G., J. E. GARNETT, AR’D T. J. CICERO. 1974. Analgesia and hyperreactivity produced by intracranial microinjections of morphine into the periaqueductal gray matter of the rat. Bchae~. Biol. 11: 303-313. SHEARD, M. H., AND G. K. AGHAJANIAN. 1968. Stimulation of the midbrain raphe: Effect on serotonin metabolism. I. Plzar~rzacol. Exg. Thcr. 163: 425-430. SHEARD, M. H., AND A. J. ZOLOVICK. 1971. Serotonin: Release in cat brain and cerebrospinal fluid on stimulation of midbrain raphe. Brain Rcs. 26: 455-458. SNIDER, R. S., AND W. T. NEIMER. 1961. A Sfercotaxic Atlas of the Cut Brain. University of Chicago Press, Chicago. SPARKES, C. G., AND P. S. J. SPENCER. 1971. Antinociceptive activity of morphine after injection of biogenic amines in the cerebral ventricles of the conscious rat. Brit. J. Pharnlacol. 42 : 23C-241. TENEN, S. S. 1968. Antagonism of the analgesic effect of morphine and other drugs by fi-chlorphenylalanine, a serotonin depletor. PsychoPharlltacologi 12 : 278-285. Tsorr, Ii., AND C. S. JANG. 1964. Studies on the site of analgesic action of morphine by intracerebral microinjection. Sci. Silz. 13 : 1099-1109. UNGERSTEDT, U. 1971. Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol. Scmd. [Szcppl.] 367: l-48. VERRI, R. A., F. G. GRAEFF, AND A. P. CORRADO. 1968. Effect of reserpine and methyltyrosine on morphine analgesia. I+tt. J. Ncz~rophar~rtacol. 7 : 283-292. W.~Y, E. L., AND F. SHEN. 1971. Catecholamines and 5-hydroxytryptaminc. Pages 229-253 in D. H. CLOUET, Ed., Narcotic Drugs: Biochcmicnl Phmncology. Plenum Press, New York. YUNGER, L. M., AND J. A. HARVEY. 1973. Effect of lesions in the medial forebrain bundle on three measures of pain sensitivity and noise-elicited startle. J. Comb. Physiol. Psychol. 83 : 173-183. SAXI.4N
NEUROLOGY
57, 1059-1066 (1977)
RESEARCH Analgesic Brain lntraventricular
DAVID
NOTE
Stimulation in the Cat: Effect Serotonin, Norepinephrine, and Dopamine
Duu~~sso~
AFXI RONALD
~ELZACK
of
’
It is now firmly established that electrical stimulation of structures in or near the mesencephalic central gray matter produces analgesia in discrete parts of the body (2, 6, 15, 16, 18-20, 22, 23, 25). Furthermore, microinjections of morphine into the ventricles or into periventricular regions produce analgesia, whereas similar injections into a variety of other sites are ineffective (12, 13, 24, 30, 36), which suggests that the action of opiate analgesic drugs is similar to that of electrical stimulation. A synergistic effect has been demonstrated between subanalgesic doses of morphine and stimulation of the dorsal raphe nucleus in rats (29)) and Mayer and Hayes (17) showed cross-tolerance to morphine and analgesic brain stimulation. Recently Akil cf al. (4) reported antagonism to analgesic brain stimulation by naloxone, a potent narcotic antagonist. The major effects of morphine have been attributed to its interaction with aminergic pathways in the brain (14, 39). In the rat, depletion of monoaminesby tetrabenazine, reserpine, and other drugs, or specific inhibition of serotonin synthesis by p-chlorphenylalanine, antagonizes both stimulation-produced analgesia (2, 3, 16) and morphine analgesia (7, 8, 35, 39.) The dopamine receptor stimulator, apomorphine, potentiates stimulation-produced analgesia, whereas blockade of dopamine receptors by Abbreviations : DOPA-dihydroxyphenylalanine. 1 Supported by Grant A7891 from the National Research Council of Canada. Dr. Dubuisson was supported by a student fellowship from Quebec Medical Research Council. The authors thank George Nieman and Warren Soper for technical assistance. Address reprint requests to Dr. Ronald Melzack, Department of Psychology, McGill University, 1205 McGregor Avenue, Montreal, PQ, Canada H3A 1Bl. 1059 Copyright Q 1977 by AcademicPress,Inc. All rights of reproduction in any form reserved.
ISSN
0014-4586
1060
DUXUISSON
AND
MELZACK
pimozide causes its reduction (2). Akil and Mayer (3) speculated that the analgesia produced by morphine and electrical stimulation in the ventral portion of the periaqueductal gray results from activation of a neural substrate which contains at least one serotonergic synapse. Evidence in support of this is provided by studies involving lesions of the medial forebrain bundle (11, 40)) administration of biogenic amines or their precursors (2, 8, 34), and measurements of forebrain serotonin content after stimulation or destruction of the midbrain raphe nuclei (1, 27, 28, 31, 32), from which a major complement of serotonergic axons is known to originate (5, 10, 37). Furthermore, Oliveras et nl. (23) noted profound analgesia produced by stimulation of a serotonin-rich region of the caudal medulla. Although the serotonergic link implicated by Akil and Mayer could ascend to the forebrain or descend to the cord from lower brain stem structures, it has been theorized (2, 3, 15, 16, 26) that morphine and analgesic brain stimulation have an ultimate common site of action in blocking nociceptive input through the dorsal horn system, a concept based on the gate control theory of Melzack and Wall (21). Sparkes and Spencer (34) measured the strength of morphine analgesia in rats after intraventricular injections of serotonin, norepinephrine, dopamine, and DOPA (dihydroxyphenylalanine) . They found that serotonin potentiated morphine analgesia, and norepinephrine, DOPA, and dopamine reduced the effectiveness of morphine. However, Calcutt et al. (S) reported that intraventricular dopamine facilitated morphine analgesia in the rat. No previous study of stimulation-produced analgesia has utilized direct intraventricular injections of biogenic amines. The present study examines the effect on stimulation-produced analgesia of intraventricular serotonin, norepinephrine, and dopamine. The subjects were 25 female cats weighing 2.4 to 3.8 kg. Under ketamine anesthesia, insulated bipolar stainless-steel electrodes were implanted stereotactically toward a point in the nucleus raphe dorsalis which is known (22) to evoke consistent analgesia. Target coordinates, based on the atlas of Snider and Niemer (33), were P-0.5, L-0.0, V-0.5. In four animals, stainless-steel cannulae were also inserted stereotactically to a point in the right lateral ventricle, 13.0 mm anterior to the vertical interaural plane. Stimulating currents consisted of biphasic rectangular p&es of 20 to 330 pA at a frequency of 30 Hz and pulse duration of 2 ms. Animals were pretested at least 24 h before the treatment sessionto determine the current levels which would produce intense analgesia to pinch with a hemostat, with a minimum of extraneous behavior such as tremor, circling, or escape. During observation, the animals were free to move about in a 75 X 75 X go-cm chamber with a Plexiglas door. A lightweight harness and leash,
ntlnclictl ;11~(1~~c 10 a rotatiug ass~i~~hl~-. allowetl tlir aniiiials to sit, walk ahut, aud explore the observation cliaiiil)er, hut prevented then1 from lyiiig down. Current was delivered through a conmutator and flexible cord. The analgesic effect of stimulation alone was observed in six animals. On the treatment day, each animal was allowed 5 min to become accustomed to the observation cliainl~er before receiviug 0.1 ml S>h formalin solution, froni a lxessurized tramcutaneous inj&or, iii the anteriormost portion of the main pad of the right front paw. Stimulation then began, and a continuous record uf pain intensity- ratings was kept on a polygraph with the aid of a four-position switch. Pain intensity was evaluated as follows : 3-licking or biting paw; 2-holding paw off floor; l-paw resting lightly on floor, not bearing weight; O-paw pressed flat on floor, dorsiflexed, with no observable difference from opposite paw. Mean paiu intensities for 3-min intervals were calculated from the polygraph record. A group of nine animals served as controls, receiving only the formalin injection. A third group, consisting of six animals, underwent a similar testing procedure except that 5 mill before the formalin injection, morphine sulfate (0.8 lug/kg) was administered intraperitoneally, and the stimulating current remained off. Observation periods lasted 30 min. A final group of four animals received a series of four treatments spaced 2 weeks apart and assigned in random order. Treatments consisted of observation periods lasting 30 niin after injection of formalin into a forel)aw ; during alternate sessions, the injection was made into the left paw rather than the right. Fifteen minutes prior to formalin injection, one of the following substances was administered into the lateral ventricle : (i) serotoniii-creatiiiine sulfate, 200 pg ; (ii) norepinephrine bitartrate, SO pg ; (iii) dopanke hydrochloride, 60 pg ; or (iv) physiological saline solution, as a control procedure. Intraventricular injections of amines in similar concentrations have been shown (9 j to have rapid, potent physiological effects in the cat. Injections were made from disposable syringes through a suitable adaptor. All compounds were dissolved in sterile, pyrogen-free physiologic saline so that the required amount of amine was contained in 0.2 nil solution. This was washed in immediately by a second injection of 0.15 ml saline. Iii the case of controls. a siiigle injection was made of 0.35 ml saline. Brain stimulation was delievered as described above, aud pain ratings were averaged for 3-miii intervals. Each animal was observed under each of the four treatment conditions. At the termination of the experiment, the animals were killed and electrode placements (Fig. 1) were determined by standard histological techniques. The position of the cannula tip within the lateral ventricle was
1062
DUBUISSON
AND
MELZACK
Frc. 1. Electrode placements which produccd analgesia in animals receiving stimulation alone (closed circles) or stimulation plus intraventricular injections (open circles).
verified by gross dissection of the fixated cerebrum prior to sectioning of the brain stem. Figure 2a shows the change in pain intensity during a 30-min period in the control animals, as well as the effects of stimulation and morphine. Both analgesic treatments clearly reduced the average pain rating during the first lo-min period, as animals began to ignore the injected paw and to place weight upon it. Figure 2b shows that dopamine or norepinephrine given intraventricularly reduced the effectivness of analgesic brain stimulation. After administration of either of these amines, the animals did not show typical analgesia, but rather tended to avoid using the injected paw throughout most of the 30-min testing period. In addition, pinch tests at the conclusion of each observation period disclosed that all animals which had received norepinephrine, and all but one of the animals which had received dopamine, showed normal withdrawal of the forepaws despite continuing brain stimulation. The combination of serotonin and brain stimulation produced unusual disturbances of motor tone which interfered with the pain rating system. In the 15-min interval prior to the formalin injection, the animals showed only mild somnolence and motor weakness. They adopted a wide stance and attempted to lie down, but were prevented from doing so by the harness. At the onset of stimulation, however, three of four animals dis-
FIG. 2. Pain intensity ratings after injection of formalin into ,the forepaw averaged for groups of animals. a-Pain rating curves for animals that received morphine or periaqueductal gray stimulation (P.4G) in comparison to controls. b-Pain ratings for animals that received stimulation plus intraventricular norepinephrine (NOR) or dopamine (DOP) versus stimulation plus intraventricular saline (S.AL).
played characteristic bizarre, cataleptiform posturing which persisted throughout the observation period. One animal crouched, motionless, with all four paws on the floor ; another showed rigid extension of both front legs; and a third held the injected paw straight and immobile at a level above its own head. The remaining animal demonstrated rapid and clear-cut analgesia with no abnormality of muscle tone. Thus, three of four animals placed weight on the injected paw throughout most of the observation period. Firm pressure with a hemostat at ‘the end of the observation period produced minimal reaction in all four animals, which suggests that analgesia was incleed present, but confounded by the neuromuscular disturbance.
1064
DUl3UISSON
AND
MELZACK
The results indicate that intraventricular norepiucpllrinc and dopamine arc autagouistic to analgesic braiu stiniulation in the cat. The findings concerning serotonin suggest a combination of analgesia and disruption of motor tone. This picture resembles the work of Sparkes and Spencer (34) on morphine analgesia. The results of intraventricular dopamine conflict with those of Akil and Liebeskind (2), who observed in the rat a potentiation of stimulation-produced analgesia by pharmacologic agents which exert a predominant central dopaminergic influence. The mechanism of action of intraventricular dopamine may be different from that of peripherally administered L-DOPA after depletion of brain amines or from that of apomorphine (2). Although the effects of biogenic amines in the present study are consistent with the concept of a common neural substrate for morphine and central gray stimulation. no single neurotransmitter can be equated with the neural mechanisms underlying narcotic analgesia. The behavior of individual animals suggests that a continua1 balance of aminergic activity may be altered in several ways either to permit or to disrupt analgesia. In conclusion, the results show that no single aminergic neurotransmitter can be equated with the neural mechanism underlying stimulation-produced analgesia. Rather, there appears to be a balance of serotonergic and catecholaminergic influences. REFERENCES 1. AGHAJANIAN, G. K., J. A. ROSECRANS, AND M. H. SHEARD. 1967. Serotonin: Release in the forebrain by stimulation of midbrain raphe. S&wcc 156: 706-708. 2. AI(IL, H., AND J. C. LIEBESKIND. 1975. Monoaminergic mechanisms of stimulationproduced analgesia. Brain Rcs. 94 : 279-296. 3. AKIL, H., AND D. J. MAYER. 1972. Antagonism of stimulation-produced analgesia by p-CPA, a serotonin synthesis inhibitor. Brain Rcs. 44 : 692-697. 4. AKIL, H., D. J. MAYER, AND J. C. LIEBESKIND. 1976. Reduction of stimulationproduced analgesia by the narcotic antagonist, naloxone. Sciolce 191: 961-962. 5. ANDEN, N. E., A. DAHLSTROM, K. FUXE, K. LARSSON, L. OLSON, AND U. UNGERSTEDT. 1966. Ascending monoamine neurons to the telencephalon and diencephalon. Acta Physiol. Stand. 67 : 313326. 6. BALAGURA, S., AND T. RALPH. 1973. The analgesic effect of electrical stimulation of the diencephalon and mesencephalon. BrailL Res. 60 : 369-379. 7. BAPAT, S. K., AND V. CHANDRA. 1968. Effect of tetrabenazine on morphine analgesia in rats. ImGun J. Plzysiol. Phanrcacol. 12 : 107-109. 8. CALCUTT, C. R., N. S. DOGGETT, AND P. S. J. SPENCER. 1971. Modification of the antinociceptive activity of morphine by centrally administered ouabain and dopamine. Psycltopharntacologia 21 : 11 l-l 17. 9. FELDBERG, W., AND S. L. SHERWOOD. 1954. Injections of drugs into the lateral ventricle of the cat. J. Pltysiol. (Land.) 123 : 148-167. 10. FUXE, K. 1965. Distribution of monoamine nerve terminals in the central nervous system. Acta PAysiol. Stand. 64, [Suppl. 2471 : 30-84.
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