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Lumbar intrathecal naloxone blocks analgesia produced by microstimulation of the ventromedial medulla in the rat
Brain Research, 236 (1982) 77-84 Elsevier Biomedical Press
77
LUMBAR INTRATHECAL NALOXONE BLOCKS ANALGESIA PRODUCED BY M I C R O S T I M U L A T I O N OF T H E V E N T R O M E D I A L M E D U L L A I N T H E RAT
GREG ZORMAN, GLYN BELCHER, JOHN E. ADAMS and HOWARD L. FIELDS Departments of Neurological Surgery, (G.B. and H.L.F.) Neurology, and Physiology, School of Medicine, University of California, San Francisco, CA 94143 (U.S.A.)
(Accepted July 16th, 1981) Key words: analgesia - - pain - - stimulation-produced analgesia - - brain stem - - tail-flick - - enkephalin - - spinal cord - - intrathecal - - naloxone
SUMMARY In lightly barbiturate-anesthetized rats, low threshold (~< 10 /~A) electrical stimulation within the rostral ventromedial medulla inhibited the tail-flick ~esponse to noxious heat. Naloxone applied intrathecally at the lumbar level revetsed this inhibition, but the same dose of naloxone applied to the cervical intrathecal space had no effect. Doses of naloxone 2 - t o 4-fold greater than the intrathecal dose did not antagonize tail-flick suppression when given systemically. Because neither systemic nor intrathecal naloxone had any effect on base-line tail-flick latencies, we conclude that the inhibition of the tail-flick response resulting from microstimulation in the ventromedial medulla is mediated by a spinal opioid synapse.
INTRODUCTION Brain stem connections to the spinal cord contribute significantly to the modulation of pain transmission. O f particular importance for analgesia is the rostral ventromedial medulla (VMM) that includes the nucleus raphe magnus and the nucleus reticularis paragigantocellularis. Electrical stimulation of the V M M produces analgesia 1,22,36 and inhibits dorsal horn nociceptive neurons3,5,10,11,1a, z6. Axons from V M M neurons project directly to superficial layers of the spinal cord dorsal horn that contain both second order pain-transmission cells and the terminals of nociceptive primary afferentsa,4,6-sA4,18-21,30. The pharmacology of the antinociceptive effect of activity in V M M neurones is as yet unclear, but the involvement of endogenous opioid peptides such as the enke0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
78 phalins is supported by the observation that the opiate antagonist naloxone reverses stimulation-produced analgesia (SPA) from the VMM in cats22, 23 and rats TM. In addition, high levels of enkephalin-like immunoreactivity have been demonstrated in terminals and perikarya of both the VMM and superficial layers of the dorsal horn 1517,27.
We recently reported that the analgesia produced by low intensity (~< l 0 / t A ) electrical stimulation restricted to the VMM was naloxone reversible 36. Because naloxone was given systemically, it was not possible to determine where in the central nervous system analgesia was blocked. One possibility is that naloxone acted at the level of the spinal cord where high levels of opiate receptor are found 2. To study more directly the role of opioid-mediated mechanisms at the level of the spinal cord, we have examined the effect of lumbar intrathecal naloxone on inhibition of the tail-flick reflex produced by microstimulation within the VMM. Our results are consistent with the hypothesis that the effect is mediated by a spinal opioid synapse. MATERIALS A N D METHODS
Experiments were performed on 14 male Sprague-Dawley rats weighing between 300 and 340 g. All rats were implanted with lumbar subarachnoid catheters (PE-10 tubing) using a modification of the technique described by Yaksh and Rudy 35. Rats were allowed to recover for at least 7 days before further experimentation, and those that developed a postsurgical neurologic deficit were not used for experiments. The VMM was stimulated using techniques described previously 36. In brief, rats were anesthetized with an initial dose of 50 mg/kg pentobarbital intraperitoneally, which was supplemented with 10 mg/kg as needed. A stainless-steel microelectrode with a 1 #m tip and a 125 # m shank diameter was positioned in the VMM using an anterior-posterior coordinate o f - - 2 . 5 , lateral 0 and 1, and vertical - - 6 to --724. After the effects of the initial dose of anesthetic had subsided, a stable baseline tail-flick latency of approximately 4 s was obtained. Tail-flick latency to noxious heat from a focused light source of constant intensity was used as the measure of analgesia. Stimulus parameters consisted of 50 Hz, 400 #s, continuous, square-pulse trains of monopolar, cathodal current applied for 5 s before and during application of noxious heat. Rats were considered analgesic only if there was no tail-flick elicited by the application of noxious heat before the 10 s cut-off. The minimum current necessary to produce inhibition of the tail-flick response to cut-offwas then determined. At no time during the course of an experiment was the intensity of focused light changed. Naloxone (15-25 #g) was administered to the lumbar subarachnoid space in volumes of 3-5 #1 via the intrathecal catheter. Following naloxone injection, the catheter was flushed through with 8-10 /~1 of balanced ion solution prepared according to the formula of Yaksh and Rudy 33. Control injections of solvent alone assured that any effects seen were due to naloxone; injection of solvent alone did not reverse analgesia. At the end of an experiment, the stimulation site was marked by passing direct current and perfusing rats with 1 ~ potassium ferrocyanidein a 10~o formaldehyde solution. Sites of stimulation were determined from Nissl-stained sections of the brain
79
Fig. 1. Reconstruction of sites within ventromedial medulla from which stimulation-produced analgesia was reversed by intrathecal lumbar naloxone, g, genu of the facial nerve; PT, pyramidal tract; IV V, fourth ventricle; VII, facial nerve nucleus.
stem. The exact position, patency, and integrity o f all spinal catheters was confirmed by dissection after sacrifice. RESULTS The sites at which stimulation with 5-10 ~ A o f current produced analgesia are shown in Fig. 1 ; all are within the V M M . In every case analgesia lasted only for the duration o f stimulation. Table I summarizes the effects o f lumbar intrathecal injection o f naloxone on SPA. Naloxone had no effect on baseline tail-flick latencies, but it consistently reversed SPA (Figs. 2 and 3). SPA was reversed approximately 10 min after injection and did not return for approximately 35 min. The naloxone effect was dose-dependent; 15 # g o f naloxone produced only partial reversal, while 25 /~g produced complete reversal. A t the time of its peak effect, 25/zg o f naloxone applied to the lumbar cord produced a significantly greater blockade o f SPA than 15/~g (Table I); the difference was significant at the P ~< 0.05 level (Student's t-test). To test for possible effects resulting from the systemic absorption o f naloxone, the jugular veins o f 5 rats were catheterized. Injection o f 2-4 times the effective lumbar intrathecal dose did not reverse SPA (Table II). Fig. 3 illustrates a typical experiment.
TABLE I Mean tail-flick latencies with and without medullary stimulation and the effect o f administration o f lumbar intrathecal naloxone (units in s)
Values represent mean tail-flick latencies and standard errors of the mean. Lumbar (15 I~g) ( n = 6)
Lumbar (25 I~g) (n = 6)
Baseline
V M M stimulation
Baseline
V M M stimulation
10
3.4 4- 0.3
10
5.5 ! 0.7*
3.2 4- 0.4
3.7 4- 0.3*
Tail-flick latency 5 min before naloxone 3.7 i 0.3 Tail-flick latency at maximum naloxone effect 3.3 4- 0.2 * Student's t-test = 2.42; P <~ 0.05.
80
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Fig. 2. A, B: effect of intrathecal cervical and lumbar naloxone on the prolongation of tail-flick latency produced by medullary stimulation. BIS, basic ion solution.
To ensure that effects seen after injection of naloxone into the lumbar subarachnoid space were not due to upward diffusion of the drug to supraspinal sites, the cervical subarachnoid space was catheterized in 4 rats by passing PE-10 tubing 2.0 cm caudally from the incision in the atlanto-occipital membrane. Doses of naloxone that reversed SPA when administered at the lumbar cord level had no effect when applied at the cervical cord level (see Table 1II). In 2 rats, naloxone was applied to the cervical cord first and then to the lumbar cord (Fig. 2A). In the others, the drug was applied initially to the lumbar cord (Fig. 2B). The effects were independent of the order of injection and, at the doses used, only the lumbar injections reversed SPA.
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Fig. 3. Effect of intrathecal lumbar and systemic naloxone on the prolongation of tail-flick latency produced by medullary stimulation.
81 TABLE II Mean tail-flick latency with and without medullary stimulation and the effect o f administration o f intravenous and lumbar intrathecal naloxone Systemic (n = 5)
Lumbar (n = 5)
Baseline
V M M stimulation
Baseline
V M M stimulation
10
3.6 4- 0.2
10
10
3.6 -+- 0.3
5.4 :k 0.9
Tail-flick latency 5 min before naloxone 3.7 4- 0.3 Tail-flick latency at maximum naloxone effect 3.2 :k 0.4 TABLE III
Medullary stimulation and the effect o f administration o f cervical and lumbar intrathecal naloxone: double-catheterized rats Cervical (n = 4)
Lumbar (n = 4)
Baseline
I / M M stimulation
Baseline
V M M stimulation
10
3.7 :k 0.4
10
10
4.0 4- 0.3
3.9 4- 0.8
Tail-flick latency 5 min before naloxone 3.9 nk 0.1 Tail-flick latency at maximum naloxone effect 3.6 :k 0.2
DISCUSSION We previously demonstrated that analgesia resulting from low-intensity (<~ 10 /~A) electrical stimulation of the V M M is antagonized by systemic naloxone 36. In this study we extended that observation by showing that naloxone restricted to the lumbar spinal cord antagonizes SPA generated from sites within the VMM. This indicates that naloxone can act locally at the level of the lumbar cord. Yaksh and Rudy showed that the bulk of naloxone remains confined to within 2 cm of a lumbar intrathecal injection site 35. However, naloxone is very lipophilic and small amounts of tritiated naloxone are found in forebrain homogenates up to 30 min after administration of radiolabeled naloxone onto the lumbar spinal cord 2s. In this study we found that doses of naloxone active at the lumbar cord did not reverse SPA when applied to the cervical cord. This suggests that the naloxone effect is not caused by rostral diffusion either intraparenchymally or within the CSF. However, because the concentration of naloxone at its site of action in the spinal cord could be very high, the possibility of nonspecific drug effects must be considered. Other workers have used similar doses of naloxone applied directly onto the cord in awake animals and no general nonspecific effects on behavior have been reported 82,33. Furthermore, the dose-dependent nature of the effect of naloxone in the present experiments would mitigate against this. However, high doses of naloxone have been shown to block some actions of G A B A lz, which is also present in high concentrations
82 in the superficial layers of the dorsal horn. On the other hand, there is no compelling evidence that G A B A has an antinociceptive action and thus any effect of naloxone via G A B A receptors, while a possibility, seems unlikely. The present results are consistent with the hypothesis that stimulation within the V M M inhibits pain transmission by a mechanism that involves the release of endogenous opioids at the level of the spinal cord. The observation that naloxone partially antagonizes inhibition of dorsal horn cells produced by V M M stimulation in rat 25 is also consistent with this idea, although conflicting data have been published 9. The dense concentration of enkephalinergic terminals and opiate receptors in the superficial layers of the dorsal horn provides a possible anatomical substrate for opioid-mediated analgesia. Observations that direct application of opiates to the spinal cord produces analgesia in animals 31,32,34 and humans 29 suggest that spinal cord opiate receptors have a role in analgesia. Although the circuitry of the superficial dorsal horn relevant to analgesia is unclear, Duggan et al. have shown that opiates are particularly effective for blocking nociceptive input to dorsal horn neurons when injected into the substantia gelatinosa lo. If V M M inhibition of the tail-flick reflex is opioid-mediated, enkephalin must be considered as a possible transmitter. Enkephalin-containing terminals in laminae I (marginal zone) and II (substantia gelatinosa) of the dorsal horn may arise from either intrinsic interneurons or medullospinal neurons. Thus, lumbar intrathecal naloxone could act by blocking the action of enkephalin released from either descending axons of V M M origin or interneurons intrinsic to the superficial dorsal horn. With the evidence currently available, it is difficult to choose between these two possible mechanisms. ACKNOWLEDGEMENTS This research was supported in part by N I D A Grant DA 01949 and N I M H Grant 5T32MH1509-04. The authors thank Gilberto Martinez for his technical assistance, Beverly J. Hunter for preparation of the manuscript, and Neil Buckley for editorial assistance. REFERENCES 1 Akaike, A., Shibata, T., Satoh, M. and Takagi, H., Analgesia induced by microinjection of morphine into and electrical stimulation of the nucleus reticularis paragigantocellularis of rat medulla oblongata, Neuropharmacology, 17 (1978) 775-778. 2 Atweh, S. F. and Kuhar, M. L, Autoradiographic localization of the opiate receptors in rat brain. I. Spinal cord and lower medulla, Brain Research, 124 (1977) 53-68. 3 Basbaum, A. I., Clanton, C. H. and Fields, H. L., Opiate and stimulus-produced analgesia: functional anatomy of a medullospinal pathway, Proc. nat. Acad. Sci. (U.S.A.), 73 (1976) 4685-4688. 4 Basbaum, A. I., Clanton, C. H. and Fields, H. L., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems, J. comp. NeuroL, 178 (1978) 209-224. 5 Beale, J. E., Martin, R. F., Applebaum, A. E. and Willis, W. D., Inhibition of primate spinothalamic tract neurons by stimulation in the region of nucleus raphe magnus, Brain Research, 114 (1976) 328-333. 6 Cervero, F., Molony, V. and Iggo, A., Extracellular and intracellular recordings from neurons in substantia gelatinosa Rolandi, Brain Research, 136 (1977) 565-569.
83 7 Christensen, B. N. and Perl, E. R., Spinal neurons specifically excited by noxious or thermal stimuli: marginal zone of the dorsal horn, J. Neurophysiol., 33 (1970) 293-307. 8 Dalstr/Sm, A. and Fuxe, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons, Acta physiol, scand., Suppl. 232 (1965) 1-55. 9 Duggan, A. W. and Griersmith, B. T., Inhibition of the spinal transmission of nociceptive information by supraspinal stimulation in the cat, Pain, 6 (1979) 149-161. 10 Duggan, A. W., Hall, J. G. and Headly, P. M., Morphine, enkephalin and the substantia gelatinosa, Nature (Lond.), 264 (1976) 456~58. I 1 Fields, H. L., Basbaum, A. I., Clanton, C. H. and Anderson, S. D., Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons, Brain Research, 126 (1977) 441454. 12 Gruol, D. L., Barker, J. L. and Smith, T. G., Naloxone antagonism of GABA-evoked membrane polarizations in cultured mouse spinal cord neurons, Brain Research, 198 (1980) 323-332. 13 Guilbaud, G., Oliveras, J. L., Giesler, G. J. and Besson, J. M., Effects induced by stimulation of the centralis inferior nucleus of the raphe on dorsal horn interneurons in cat's spinal cord, Brain Research, 126 (1977) 355-360. 14 Hentall, I. D. and Fields, H. L., Segmental and descending influences on intraspinal thresholds of single C-fibres, J. NeurophysioL, 42 (1979) 1527-1537. 15 Hokfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalinimmunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 16 Hokfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. nat. Acad. Sci. (U.S.A.), 74 (1977) 3081-3085. 17 Hokfelt, T., Terenius, L., Kuypers, H. G. J. M. and Dann, O., Evidence for enkephalin immunoreactive neurons in the medulla oblongata projecting to the spinal cord, Neurosci. Lett., 4 (1979) 55-60. 18 Light, A. R. and Perl, E. R., Reexamination of the dorsal root projection of the spinal dorsal horn, including observations on the differential termination of coarse and fine fibers, J. comp. Neurol., 186 (1979) 117-131. 19 Light, A. R. and Perl, E. R., Differential termination of large-diameter and small-diameter primary afferent fibers in the spinal dorsal gray matter as indicated by labeling with horseradish peroxidase, Neurosci. Lett., 6 (1977) 59-63. 20 Light, A. R., Tervino, D. C. and Perl, E. R., Morphological features of functionally identified neurons in the marginal zone and substantia gelatinosa of the spinal dorsal horn, J. comp. Neurol., 186 (1979) 151-171. 21 Martin, R. F., Jordan, L. M. and Willis, W. D., Differential projection of cat medullary raphe nuclei in spinal cord white matter, J. comp. Neurol., 182 (1978) 77-87. 22 Oliveras, J. L., Hosobuchi, Y., Redjemi, F., Guilbaud, G. and Besson, J. M., The opiate antagonist naloxone strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior), Brain Research, 120 (1977) 221-229. 23 Oliveras, J. L., Redjemi, F., Guilbaud, G. and Besson, J. M., Analgesia induced by electrical stimulation of the inferior centralis nucleus of the raphe in the cat, Pain, 1 (1977) 139-145. 24 Pellegrino, L. J., Pellegrino, A. S. and Cushman, A. J., A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979. 25 Rivot, J. P., Chaouch, A. and Besson, J. M., The influence of naloxone on C fiber response of dorsal horn neurons and their inhibitory control by raphe magnus stimulation, Brain Research, 176 (1979) 355-364. 26 Rivot, J. P., Chaouch, A. and Besson, J. M., Effects of naloxone on the inhibitory action induced by stimulation of the raphe magnus on responses of spinal cord dorsal horn cells, Neurosci. Lett., Suppl. 1 (1978) $324. 27 Sar, M., Stumpf, W. E., Miller, R. J., Chang, K. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. comp. Neurol., 182 (1978) 17-37. 28 Tang, A. H. and Schoenfeld, M. J., Comparison of subcutaneous and spinal arachnoid injections of morphine and naloxone on analgesic tests in the rat, Europ. J. Pharmacol., 52 (1978) 215-223. 29 Wang, J. K., Nauss, L. E. and Thomas, J. E., Pain relief by intrathecally applied morphine in man, Anesthesiology, 50 (1979) 149-151. 30 Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., The somatotopic organization of the nucleus raphe magnus and surrounding brain stem structures as revealed by HRP slow-release gels, Brain Research, 151 (1980) 1-15.
84 31 Yaksh, T. L., Analgesic actions of intrathecal opiates in cat and primates, Brain Research, 153 (1978) 205-210. 32 Yaksh, T. L. and Rudy, T. A., Analgesia mediated by a direct spinal action of narcotics, Science, 192 (1976) 1357-1358. 33 Yaksh, T. L. and Rudy, T. A., Studies on the direct spinal action of narcotics in the production of analgesia in the rat, J. Pharmacol. exp. Ther., 202 (1977) 411-428. 34 Yaksh, T. L. and Rudy, T. A., Narcotic analgesics : CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359. 35 Yaksh, T. L. and Rudy, T. A., Chronic catherization of the spinal arachnoid space, Physiol. Behav., 17 (1976) 1031-1036. 36 Zorman, G., Hentall, I. D., Adams, J. E. and Fields, H. L., Naloxone-reversible analgesia produced by microstimulation in the rat medulla, Brain Research, 219 (1981) 137-148.
77
LUMBAR INTRATHECAL NALOXONE BLOCKS ANALGESIA PRODUCED BY M I C R O S T I M U L A T I O N OF T H E V E N T R O M E D I A L M E D U L L A I N T H E RAT
GREG ZORMAN, GLYN BELCHER, JOHN E. ADAMS and HOWARD L. FIELDS Departments of Neurological Surgery, (G.B. and H.L.F.) Neurology, and Physiology, School of Medicine, University of California, San Francisco, CA 94143 (U.S.A.)
(Accepted July 16th, 1981) Key words: analgesia - - pain - - stimulation-produced analgesia - - brain stem - - tail-flick - - enkephalin - - spinal cord - - intrathecal - - naloxone
SUMMARY In lightly barbiturate-anesthetized rats, low threshold (~< 10 /~A) electrical stimulation within the rostral ventromedial medulla inhibited the tail-flick ~esponse to noxious heat. Naloxone applied intrathecally at the lumbar level revetsed this inhibition, but the same dose of naloxone applied to the cervical intrathecal space had no effect. Doses of naloxone 2 - t o 4-fold greater than the intrathecal dose did not antagonize tail-flick suppression when given systemically. Because neither systemic nor intrathecal naloxone had any effect on base-line tail-flick latencies, we conclude that the inhibition of the tail-flick response resulting from microstimulation in the ventromedial medulla is mediated by a spinal opioid synapse.
INTRODUCTION Brain stem connections to the spinal cord contribute significantly to the modulation of pain transmission. O f particular importance for analgesia is the rostral ventromedial medulla (VMM) that includes the nucleus raphe magnus and the nucleus reticularis paragigantocellularis. Electrical stimulation of the V M M produces analgesia 1,22,36 and inhibits dorsal horn nociceptive neurons3,5,10,11,1a, z6. Axons from V M M neurons project directly to superficial layers of the spinal cord dorsal horn that contain both second order pain-transmission cells and the terminals of nociceptive primary afferentsa,4,6-sA4,18-21,30. The pharmacology of the antinociceptive effect of activity in V M M neurones is as yet unclear, but the involvement of endogenous opioid peptides such as the enke0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
78 phalins is supported by the observation that the opiate antagonist naloxone reverses stimulation-produced analgesia (SPA) from the VMM in cats22, 23 and rats TM. In addition, high levels of enkephalin-like immunoreactivity have been demonstrated in terminals and perikarya of both the VMM and superficial layers of the dorsal horn 1517,27.
We recently reported that the analgesia produced by low intensity (~< l 0 / t A ) electrical stimulation restricted to the VMM was naloxone reversible 36. Because naloxone was given systemically, it was not possible to determine where in the central nervous system analgesia was blocked. One possibility is that naloxone acted at the level of the spinal cord where high levels of opiate receptor are found 2. To study more directly the role of opioid-mediated mechanisms at the level of the spinal cord, we have examined the effect of lumbar intrathecal naloxone on inhibition of the tail-flick reflex produced by microstimulation within the VMM. Our results are consistent with the hypothesis that the effect is mediated by a spinal opioid synapse. MATERIALS A N D METHODS
Experiments were performed on 14 male Sprague-Dawley rats weighing between 300 and 340 g. All rats were implanted with lumbar subarachnoid catheters (PE-10 tubing) using a modification of the technique described by Yaksh and Rudy 35. Rats were allowed to recover for at least 7 days before further experimentation, and those that developed a postsurgical neurologic deficit were not used for experiments. The VMM was stimulated using techniques described previously 36. In brief, rats were anesthetized with an initial dose of 50 mg/kg pentobarbital intraperitoneally, which was supplemented with 10 mg/kg as needed. A stainless-steel microelectrode with a 1 #m tip and a 125 # m shank diameter was positioned in the VMM using an anterior-posterior coordinate o f - - 2 . 5 , lateral 0 and 1, and vertical - - 6 to --724. After the effects of the initial dose of anesthetic had subsided, a stable baseline tail-flick latency of approximately 4 s was obtained. Tail-flick latency to noxious heat from a focused light source of constant intensity was used as the measure of analgesia. Stimulus parameters consisted of 50 Hz, 400 #s, continuous, square-pulse trains of monopolar, cathodal current applied for 5 s before and during application of noxious heat. Rats were considered analgesic only if there was no tail-flick elicited by the application of noxious heat before the 10 s cut-off. The minimum current necessary to produce inhibition of the tail-flick response to cut-offwas then determined. At no time during the course of an experiment was the intensity of focused light changed. Naloxone (15-25 #g) was administered to the lumbar subarachnoid space in volumes of 3-5 #1 via the intrathecal catheter. Following naloxone injection, the catheter was flushed through with 8-10 /~1 of balanced ion solution prepared according to the formula of Yaksh and Rudy 33. Control injections of solvent alone assured that any effects seen were due to naloxone; injection of solvent alone did not reverse analgesia. At the end of an experiment, the stimulation site was marked by passing direct current and perfusing rats with 1 ~ potassium ferrocyanidein a 10~o formaldehyde solution. Sites of stimulation were determined from Nissl-stained sections of the brain
79
Fig. 1. Reconstruction of sites within ventromedial medulla from which stimulation-produced analgesia was reversed by intrathecal lumbar naloxone, g, genu of the facial nerve; PT, pyramidal tract; IV V, fourth ventricle; VII, facial nerve nucleus.
stem. The exact position, patency, and integrity o f all spinal catheters was confirmed by dissection after sacrifice. RESULTS The sites at which stimulation with 5-10 ~ A o f current produced analgesia are shown in Fig. 1 ; all are within the V M M . In every case analgesia lasted only for the duration o f stimulation. Table I summarizes the effects o f lumbar intrathecal injection o f naloxone on SPA. Naloxone had no effect on baseline tail-flick latencies, but it consistently reversed SPA (Figs. 2 and 3). SPA was reversed approximately 10 min after injection and did not return for approximately 35 min. The naloxone effect was dose-dependent; 15 # g o f naloxone produced only partial reversal, while 25 /~g produced complete reversal. A t the time of its peak effect, 25/zg o f naloxone applied to the lumbar cord produced a significantly greater blockade o f SPA than 15/~g (Table I); the difference was significant at the P ~< 0.05 level (Student's t-test). To test for possible effects resulting from the systemic absorption o f naloxone, the jugular veins o f 5 rats were catheterized. Injection o f 2-4 times the effective lumbar intrathecal dose did not reverse SPA (Table II). Fig. 3 illustrates a typical experiment.
TABLE I Mean tail-flick latencies with and without medullary stimulation and the effect o f administration o f lumbar intrathecal naloxone (units in s)
Values represent mean tail-flick latencies and standard errors of the mean. Lumbar (15 I~g) ( n = 6)
Lumbar (25 I~g) (n = 6)
Baseline
V M M stimulation
Baseline
V M M stimulation
10
3.4 4- 0.3
10
5.5 ! 0.7*
3.2 4- 0.4
3.7 4- 0.3*
Tail-flick latency 5 min before naloxone 3.7 i 0.3 Tail-flick latency at maximum naloxone effect 3.3 4- 0.2 * Student's t-test = 2.42; P <~ 0.05.
80
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J 100
Fig. 2. A, B: effect of intrathecal cervical and lumbar naloxone on the prolongation of tail-flick latency produced by medullary stimulation. BIS, basic ion solution.
To ensure that effects seen after injection of naloxone into the lumbar subarachnoid space were not due to upward diffusion of the drug to supraspinal sites, the cervical subarachnoid space was catheterized in 4 rats by passing PE-10 tubing 2.0 cm caudally from the incision in the atlanto-occipital membrane. Doses of naloxone that reversed SPA when administered at the lumbar cord level had no effect when applied at the cervical cord level (see Table 1II). In 2 rats, naloxone was applied to the cervical cord first and then to the lumbar cord (Fig. 2A). In the others, the drug was applied initially to the lumbar cord (Fig. 2B). The effects were independent of the order of injection and, at the doses used, only the lumbar injections reversed SPA.
® I0 r - O - - O - O
8/
0
0--0
VMM stimulation
D
\
o
/
N
o ¢
I
o
10
I
20 A A 50,ug lO,ul Naloxone 81S I.V. lumbar
I
I
30
40
A 152Jg Naloxone lumbar
I
I
50 6o Time (min)
Fig. 3. Effect of intrathecal lumbar and systemic naloxone on the prolongation of tail-flick latency produced by medullary stimulation.
81 TABLE II Mean tail-flick latency with and without medullary stimulation and the effect o f administration o f intravenous and lumbar intrathecal naloxone Systemic (n = 5)
Lumbar (n = 5)
Baseline
V M M stimulation
Baseline
V M M stimulation
10
3.6 4- 0.2
10
10
3.6 -+- 0.3
5.4 :k 0.9
Tail-flick latency 5 min before naloxone 3.7 4- 0.3 Tail-flick latency at maximum naloxone effect 3.2 :k 0.4 TABLE III
Medullary stimulation and the effect o f administration o f cervical and lumbar intrathecal naloxone: double-catheterized rats Cervical (n = 4)
Lumbar (n = 4)
Baseline
I / M M stimulation
Baseline
V M M stimulation
10
3.7 :k 0.4
10
10
4.0 4- 0.3
3.9 4- 0.8
Tail-flick latency 5 min before naloxone 3.9 nk 0.1 Tail-flick latency at maximum naloxone effect 3.6 :k 0.2
DISCUSSION We previously demonstrated that analgesia resulting from low-intensity (<~ 10 /~A) electrical stimulation of the V M M is antagonized by systemic naloxone 36. In this study we extended that observation by showing that naloxone restricted to the lumbar spinal cord antagonizes SPA generated from sites within the VMM. This indicates that naloxone can act locally at the level of the lumbar cord. Yaksh and Rudy showed that the bulk of naloxone remains confined to within 2 cm of a lumbar intrathecal injection site 35. However, naloxone is very lipophilic and small amounts of tritiated naloxone are found in forebrain homogenates up to 30 min after administration of radiolabeled naloxone onto the lumbar spinal cord 2s. In this study we found that doses of naloxone active at the lumbar cord did not reverse SPA when applied to the cervical cord. This suggests that the naloxone effect is not caused by rostral diffusion either intraparenchymally or within the CSF. However, because the concentration of naloxone at its site of action in the spinal cord could be very high, the possibility of nonspecific drug effects must be considered. Other workers have used similar doses of naloxone applied directly onto the cord in awake animals and no general nonspecific effects on behavior have been reported 82,33. Furthermore, the dose-dependent nature of the effect of naloxone in the present experiments would mitigate against this. However, high doses of naloxone have been shown to block some actions of G A B A lz, which is also present in high concentrations
82 in the superficial layers of the dorsal horn. On the other hand, there is no compelling evidence that G A B A has an antinociceptive action and thus any effect of naloxone via G A B A receptors, while a possibility, seems unlikely. The present results are consistent with the hypothesis that stimulation within the V M M inhibits pain transmission by a mechanism that involves the release of endogenous opioids at the level of the spinal cord. The observation that naloxone partially antagonizes inhibition of dorsal horn cells produced by V M M stimulation in rat 25 is also consistent with this idea, although conflicting data have been published 9. The dense concentration of enkephalinergic terminals and opiate receptors in the superficial layers of the dorsal horn provides a possible anatomical substrate for opioid-mediated analgesia. Observations that direct application of opiates to the spinal cord produces analgesia in animals 31,32,34 and humans 29 suggest that spinal cord opiate receptors have a role in analgesia. Although the circuitry of the superficial dorsal horn relevant to analgesia is unclear, Duggan et al. have shown that opiates are particularly effective for blocking nociceptive input to dorsal horn neurons when injected into the substantia gelatinosa lo. If V M M inhibition of the tail-flick reflex is opioid-mediated, enkephalin must be considered as a possible transmitter. Enkephalin-containing terminals in laminae I (marginal zone) and II (substantia gelatinosa) of the dorsal horn may arise from either intrinsic interneurons or medullospinal neurons. Thus, lumbar intrathecal naloxone could act by blocking the action of enkephalin released from either descending axons of V M M origin or interneurons intrinsic to the superficial dorsal horn. With the evidence currently available, it is difficult to choose between these two possible mechanisms. ACKNOWLEDGEMENTS This research was supported in part by N I D A Grant DA 01949 and N I M H Grant 5T32MH1509-04. The authors thank Gilberto Martinez for his technical assistance, Beverly J. Hunter for preparation of the manuscript, and Neil Buckley for editorial assistance. REFERENCES 1 Akaike, A., Shibata, T., Satoh, M. and Takagi, H., Analgesia induced by microinjection of morphine into and electrical stimulation of the nucleus reticularis paragigantocellularis of rat medulla oblongata, Neuropharmacology, 17 (1978) 775-778. 2 Atweh, S. F. and Kuhar, M. L, Autoradiographic localization of the opiate receptors in rat brain. I. Spinal cord and lower medulla, Brain Research, 124 (1977) 53-68. 3 Basbaum, A. I., Clanton, C. H. and Fields, H. L., Opiate and stimulus-produced analgesia: functional anatomy of a medullospinal pathway, Proc. nat. Acad. Sci. (U.S.A.), 73 (1976) 4685-4688. 4 Basbaum, A. I., Clanton, C. H. and Fields, H. L., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems, J. comp. NeuroL, 178 (1978) 209-224. 5 Beale, J. E., Martin, R. F., Applebaum, A. E. and Willis, W. D., Inhibition of primate spinothalamic tract neurons by stimulation in the region of nucleus raphe magnus, Brain Research, 114 (1976) 328-333. 6 Cervero, F., Molony, V. and Iggo, A., Extracellular and intracellular recordings from neurons in substantia gelatinosa Rolandi, Brain Research, 136 (1977) 565-569.
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