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Effects of locus coeruleus lesions on morphine-induced antinociception
Brain Research, 188 (1980) 79-91 © Elsevier/North-HollandBiomedicalPress
79
EFFECTS OF LOCUS COERULEUS LESIONS ON MORPHINE-INDUCED ANTINOCICEPTION
DONNA L. HAMMOND and HERBERTK. PROUDFIT University of Illinois, Department of Pharmacology, College of Medicine, Chicago, Ill. 60680 (U. S..4.)
(Accepted June 9th, 1979) Key words: antinociception -- locus coeruleus -- morphine -- norepinephrine -- ventral para-
brachial nucleus
SUMMARY These studies were designed to examine the role of the norepinephrine-containing cells comprising the nucleus locus coeruleus (LC) in the mediation of pain perception and morphine-induced antinociception. Nociceptive threshold and morphine-induced analgesia were measured following monosodium-L-glutamate lesions of the LC and adjacent tegmentum (nucleus parabrachialis ventralis; PBV) at 17, 24 and 31 days after surgery. Nociceptive thresholds assessed by the tail flick and hot plate assays were not altered following lesions which included both the LC and PBV (Group l) or by lesions of the PBV (Group 2) alone. Examination of lesion-induced effects on the capacity of morphine to induce analgesia revealed that damage which included both LC and PBV as well as that confined primarily to the PBV resulted in attenuation of analgesia induced by morphine. Those lesions which involved the LC altered norepinephrine content in the cortex, spinal cord and medial brain stem; however, no correlation could be demonstrated between the attenuation of morphine-induced analgesia and the changes in norepinephrine content of the brain regions examined. Thus, destruction of the LC does not appear to be responsible for the decreased effectiveness of morphine. The only region consistently damaged in both groups 1 and 2 was the ventral parabrachial nucleus. Therefore, we tentatively conclude that destruction of the PBV was responsible for the observed attenuation of morphine analgesia.
INTRODUCTION The participation of brain norepinephrine (NE) systems in the mediation of morphine analgesia has been suggested by several lines of pharmacological evidence. However, the precise role of NE is obscured by the contradictory results of these
80 studies. For example, depletion of whole brain N E by various agents has been reported to antagonize~,4,6, '~4, potentiateg,~o, 40 or exert no effect 13 on morphineinduced analgesia. These contradictory results may in part be accounted for by differences in species, analgesiometric tests, doses and pretreatment schedules utilized. However, it is also likely that complex mechanisms are involved in the relationship between NE systems and those mediating morphine analgesia; some perhaps facilitating and others opposing the actions of morphine. Thus, studies of discrete NEcontaining nuclei might be expected to yield less equivocal results than those using pharmacological agents which produce general and indiscriminant alterations in whole brain NE levels. One such NE-containing nucleus, the nucleus locus coeruleus (LC), has been suggested to be involved in the expression of morphine-induced analgesia. For example, cells in the LC respond to noxious peripheral stimulation with a marked, though transient, increase in firing 19. In addition, the systemiO ,~ or iontophoretic 5 application of morphine to these cells produces a naloxone-reversible depression of firing. The sensitivity of the LC neurons to morphine is not unexpected in view of the high density of opiate receptors it possesses 2,31,3z. Morphine has also been suggested to inhibit the release of NE from axon terminals originating in the LCa2; a similar action of morphine has been demonstrated in cortical slices 26. A role for the LC in the actions of opiates has also been suggested by anatomical studies demonstrating that the LC is the origin of a diffuse network of projections to the cerebral, hippocampal and cerebellar corticeslS,'~2, 29,a:3,37,5°,52 in addition to various subcortical areaslS,"2,2a,aT, '~2. A salient feature of these projections is their presence in areas known to be involved in the mediation of morphine analgesia such as the periaqueductal gray z3,a7, n. raphe dorsalis 23,36 and n. raphe magnus it. Recently, a direct coeruleo-spinal projection has been described ~7,4~, a p~rtion of which terminates in the superficial layers of the dorsal horn. Furthermore, lesions of the LC and its projections have been shown to alter morphine-induced analgesia. For example, 6-hydroxydopamine-induced lesions of the dorsal noradrenergic bundle (DNB) have been reported to potentiate morphine analgesia, although destruction of neither the dorsal nor the ventral bundle altered sensitivity to pain 34. Since the LC has been identified as the principal source of fibers ill the DNB 22,5°,52, these data suggest that the LC may have an inhibitory influence on systems that mediate morphine-induced analgesia. However, other studies using electrolytic lesions of the LC have reported increases in nociceptive threshold 8,43 and attenuation of morphine analgesia "°,~3 assessed by flinch jump and compression tests. The interpretation of these latter findings is somewhat difficult since electrolytic lesions damage axons of passage and blood vessels in addition to cell perikarya in the target area. Thus, damage to adjacent fiber tracts may contribute to the reported effect of LC lesions and may account for the different results obtained using electrolytic and 6OH DA-induced lesions. The present studies were designed to circumvent these problems by investigating the effects of LC destruction on nociceptive threshold and morphine-induced analgesia using lesions produced by the local injection of monosodium-L-glutamate. This
81 method has been reported to spare axons of passage, blood vessels and glia while producing destruction of cell perikarya restricted to the area of injection 28,47,48,53. In addition, previous studies only measured changes in forebrain NE levels following LC lesions. However, the LC also projects to the medial brain stem 11 and spinal cord dorsal horn 27,44, sites at which the local application of morphine has been shown to induce analgesia1,21,35, ~5. For this reason, we examined the effect of LC lesions on N E content in the spinal cord, cortex and the medial brain stem in an attempt to ascertain the existence of correlations between lesion-induced changes in NE levels in these areas and changes in morphine-induced analgesia. METHODS
Lesioning procedure Female Sprague-Dawley-derived rats weighing between 220 and 280 g were used for these studies. The animals were anesthetized with sodium pentobarbital, placed in a stereotaxic frame, and the tip of a 28-gauge stainless steel injection cannula was stereotaxically positioned in the locus coeruleus. The injection cannula was inclined at a 20 ° angle to avoid rupturing the superior sagittal sinus. The injection system consisted of a 40 cm length of polyethylene (PE-20) tubing connecting the injection cannula with a 5/zl Hamilton syringe which was driven by an infusion pump. Bilateral lesions were made by injecting 5 #1 of 1 ~ monosodium-L-glutamate in saline at a rate of 0.59 /A/rain into each of 4 sites within the LC (two injections per side). The stereotaxic coordinates for these injection sites were as follows: P 1.6, H 1.8, L ± 1.3 m m and P 1.2, H 2.0, L --4- 1.3 m m with the incisor bar at + 5 . 0 mm. The injection of the glutamate solution was monitored by observing the movement of an air bubble in the tubing. The cannula was left in place for an additional 2 min to minimize back diffusion when it was withdrawn. The animals were housed in groups of 5 and allowed 17 days to recover from the effects of surgery.
Analgesiometric tests Two analgesiometric tests (tail flick and hot plate) were utilized to acertain alterations in nociceptive threshold and morphine analgesia. Tail flick latency (TFL) was defined as the elapsed time between onset of a high intensity light beam focused on the blackened tail and the reflex tail flick response. The average of three determinations was recorded. The number of seconds which elapsed before the rat licked its hind paw or jumped after placement on a 55 °C hot plate was defined as the hot plate latency (HPL). To avoid injury, cut-offvalues of 14 and 40 sec were chosen for the tail flick and hot plate, respectively. T F L and H P L were expressed as an antinociceptive index (AI) to minimize variability due to differences in pre-drug latencies and thus allow more valid comparisons among animals. The AI was calculated as follows: AI
=
latency after morphine - - control latency , , . An/-xl of 1.0 denotes antinociception cut-off latency - - control latency
of a magnitude equal to or greater than the appropriate cut-off latency, while an AI of 0.0 denotes absence of antinociception. Negative values indicate hyperalgesia.
82
Assessment of lesion effects Nociceptive threshold. Prior to surgery, tail flick and hot plate latencies were determined and these values were compared with those obtained 17, 24, and 31 days following surgery. Non-lesioned control animals were tested concurrently. The rats were first tested on the tail flick apparatus and then immediately tested on the hot plate. Morphine-induced analgesia. Following assessment of the nociceptive threshold on each of the three test days, the capacity of systemic morphine to induce analgesia in both lesioned and non-lesioned control animals was examined. Each group of rats received doses of 2.0, 5.0, and 10.0 mg/kg morphine sulfate (Merck) subcutaneously on days 17, 24, and 31, respectively. Drug effects were assessed 30 min following injection, the time previously determined as the peak of morphine-induced analgesia.
NE determination NE content in the cortex, lumbosacral spinal cord, and medial brain stem was determined spectrofluorometrically using a modification of the method reported by Welch and Welch 54 for extraction of the amine and the method of Chang 1° for development of the fluorophore. Following decapitation, the spinal cord was rapidly removed, all roots were dissected away and that portion of the cord caudal to and including the lumbar enlargement was assayed. The brain was quickly removed from the calvarium and a slice of cortex was dissected free of the underlying diencephalon. The brain stem was then blocked and a 2 mm thick parasagittal segment containing the raphe complex removed for assay. The regional NE content of lesioned animals was expressed as a percentage of NE levels in corresponding areas of non-lesioned animals.
Histology Histological verification of LC destruction following glutamate lesions was made in all lesioned animals. The degree of damage sustained by the LC in lesioned animals was determined by comparison with brain sections from intact control animals in which the distribution of NE-containing cells in the LC had been determined by fluorescence histochemical visualization. Identification of the lesion site in l 1 animals was based on the examination of 40 # m coronal sections taken every 200 #m and stained with cresyl violet only. In the remaining 9 animals, LC damage was determined using fluorescence histochemical methods in addition to routine histological examination. The brains from these animals were removed, the appropriate block of tissue frozen and l0 #m sections taken at 200 #m intervals through the LC. The sections were reacted with glyoxylic acid 12 to produce a NE fluorophore and examined using fluorescence microscopy. Alternate 30 #m sections of these brains were stained with cresyl violet.
Statistical analysis Post-lesion TFLs and HPLs were expressed as a percentage of pre-lesion values. Post-lesion nociceptive thresholds of lesioned animals were compared with those of
83
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Fig. 1. Camera lucida drawings illustrating the location of representative monosodium-L-glutamateinduced lesions in the dorsolateral pontine tegmentum. A: intact control; B : lesion including thelocus coeruleus and the ventral parabrachial nucleus (LC + PBV); C: lesion restricted primarily to the ventral parabrachial nucleus (PBV). Abbreviations: CER, cerebellum; el, ependymal lining; IV, fourth ventricle; LC, nucleus locus coeruleus; MeV, mesencephalic nucleus of the trigeminal; PBV, nucleus parabrachialis ventralis; SCP, superior cerebellar peduncle. intact control animals with a two-way analysis of variance with replications and N e w m a n - K e u l s test for multiple comparisons 17. The response of lesioned animals to various doses of morphine was compared to that of non-lesioned control animals by means of a two-way analysis of variance with replications and N e w m a n - K e u l s test. Spearman's Rank Correlation Coefficient 45 was utilized to assess the degree of association between regional changes in N E content and alterations in morphineinduced analgesia. RESULTS Lesion site
Glutamate-induced lesions examined in sections stained with cresyl violet appeared as cylindrical or teardrop-shaped regions of glialproliferation exhibiting marked loss of neuronal perikarya. The size of the lesion at the site of injection was
84 usually between 0.3 mm and 0.6 mm in diameter. The glutamate solution apparently spread dorsally along the cannula resulting in an elongated pattern of gliosis (Fig. 1B and C). No sign of physical damage due to the large injection volume was seen; the track produced by insertion of the injection cannula was the only observable evidence of destruction by physical forces. Fluorescence histochemical observations of sections taken through the LC revealed a reduction and in many cases a complete absence of catecholamine fluorescence with lesions localized to the LC. A given animal was considered to have received a lesion of the LC if the following criteria were met: (1) the presence o f a cannula track in the LC, (2) loss of cell bodies and increased gliosis in the LC, (3) reduction or lack of catecholamine fluorescence in the LC, and (4) reduction of cortical NE to less than 80 ~ of control values. Accordingly, 8 animals possessed bilateral lesions of the LC. In 7 of the 8 animals with bilateral LC lesions, the cannula track, gliosis, and loss of cell bodies also extended beneath the LC to the nucleus parabrachialis ventralis (PBV). Such a lesion is illustrated in Fig. 1B. The lesions in these 7 animals were considered to occupy similar sites and will be referred to as LC 4- PBV-lesioned animals. In the remaining bilaterally lesioned animal, damage was restricted to the LC only. Three animals had unilateral lesions of the LC; however, due to technical difficulties the precise extent of the lesion could not be determined. These animals were not included in any group for evaluation of lesion-induced alterations in the nociceptive threshold or morphineinduced analgesia. However, they were used to assess the relationship between alterations in regional NE content and alterations in morphine-induced analgesia. Nine animals did not have lesions of the LC. In 7 of these animals the cannula track and gliosis appeared lateral to the LC and extended ventrally to include the PBV, either unilaterally (n = 6) or bilaterally (n -- 1). Such a lesion is illustrated in Fig. 1C. These animals were grouped together and will be referred to as PBV-lesioned animals. The two remaining animals were not included in any group due to difficulty in defining the precise extent of the lesions. These animals were used to assess the relationship between alterations in NE content and alterations in morphine-induced analgesia. Regional N E content Bilateral lesions of the LC significantly reduced cortical, cord, and medial brain stem NE content to 53.03 4- 4.99~/o (n = 6), 72.82 J_ 4.80~ (n 7), and 86.74 ± 2.88 ~ (n = 3) of control (mean ± S.E.M.), respectively (Student t-test, P < 0.05, all cases). Absolute levels of NE expressed as ng/g wet weight tissue (mean ~ S.E.M.) in corresponding areas of non-lesioned control animals were 246.48 ~_ 17.35 (n = 8), 269.49 ~ 16.85 (n = 9), and 604.70 ~ 24.84 (n 4) for cortex, spinal cord and medial brain stem, respectively. Followinglesions which produced varying degrees of damage to the LC, NE levels ranged from 79.29 to 181.70 ng/g in cortex; 152.63 to 330.0 ng/g in the spinal cord; and 492.31 to 551.99 ng/g in the medial brain stem. Lesions of the PBV which spared the LC did not alter the regional NE content significantly. In these animals, cortical, cord and medial brain stem NE levels were 93.01 A 4.29)o (n ~ 7), 85.30 ~ 9.50 °/o (n = 7), and 98.21 ~ 7.98 ~ (n = 3) of control respectively (Student ttest, P ~ 0.05, all cases).
85
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Fig. 2. The effect of lesions on the dose-response relationship of morphine administered to nonlesioned control rats (0 n = 19), LC + PBV-lesioned rats (ll n = 7), and PBV-lesioned rats (A n = 7). The antinociceptive index is plotted on the ordinate and the log dose is plotted on the abscissa. Each point represents the mean AI 4- S.E.M.
Behavioral effects of lesions None of the glutamate-lesioned animals in this study exhibited any behavioral alterations or motor deficits. Animals were active throughout the duration of the experiment and exhibited normal exploratory behavior. All animals displayed normal feeding and drinking patterns accompanied by normal weight gain. These animals were essentially indistinguishable from unoperated controls.
Alterations in nocieeptive threshold The capacity of the various lesions to produce alterations in reactivity to noxious stimuli was assessed using both the tail flick and hot plate tests. The statistical significance of alterations in nociceptive threshold was determined using a two-way analysis of variance 17 which compared response latencies of the three groups of animals (LC 6- PBV, PBV, and intact control) measured at 17, 24, and 31 days following surgery. This analysis revealed no statistically significant alterations in nociceptive threshold for any of the three groups as a function of time after surgery and no differences between the intact control group and either of the lesion groups compared at the three time points.
Alterations in morphine-induced analgesia Fig. 2 illustrates the capacity of various doses of morphine to elevate the T F L and H P L in intact control, LC ÷ PBV, and PBV-lesioned animals. These data indicate that lesions of both the LC 6- PBV and the PBV resulted in potent attenuation of morphine-induced analgesia regardless of the analgesiometric test. Both the lesioned and non-lesioned control animals responded to morphine in a statistically significant dose-related fashion (two-way ANOVA, P < 0.05). However, the magnitude of the
86 Spinal
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Fig. 3. Correlation between the magnitude of antinociception (AI) induced by morphine sulfate (5 mg/kg) and NE levels in (A) spinal cord, (B) cortex, and (C) medial brain stem. Ordinate: antinociceptive index (AI) derived from tail flick latency measurements. Abscissa : NE levels in the spinal cord, cortex, and medial brain stem determined in each animal and expressed as per cent of NE in corresponding areas of intact control animals. Spearman's Rank Correlation Coefficients for spinal cord = -0.01 ; cortex = 0.09; medial brain stem = 0.04. analgesia produced by identical doses of morphine administered to both the LC + PBV and PBV-lesioned animals was significantly less than that produced in nonlesioned control animals as assessed by either HP or TF tests (Newman-Keuls, P < 0.05). Furthermore, the log dose-response curve of the LC ÷ PBV-lesioned animals was not significantly different from that of the PBV-lesioned animals ascertained by either analgesiometric test (Newman-Keuls, P > 0.05). Thus, both lesion placements were equipotent in their ability to attenuate morphine-induced analgesia.
Regional NE content and morphine analgesia The relationship between alterations in the capacity of morphine to elevate the TFL and HPL and alterations in regional NE content following lesions of the LC + PBV and PBV was also examined. Fig. 3 illustrates the correlation between NE content of the spinal cord, cortex, and medial brain stem and analgesia induced by administration of morphine sulfate (5 mg/kg) to lesioned animals using the tail flick test. Assessment of the degree of association between changes in cortical, spinal cord, or medial brain stem NE levels and the response of lesioned animals to morphine was made using Spearman's Rank Correlation CoefficienOL No significant correlation existed between these two variables for any of the brain regions examined (rs = --0.01, 0.09, and 0.04, respectively; P > 0.05). Similar results were obtained using the hot plate test. Spearman's Rank Correlation Coefficients for these data were 0.02, ~ ) . 3 3 , and 0.08 for cortical, spinal cord, and brain stem NE content versus morphine
87 analgesia, respectively. None of these coefficients were statistically significant (P > 0.05). Furthermore, no significant correlations were observed between the analgesia resulting from either 2 or l0 mg/kg of morphine and NE content in any of the areas examined. Thus, the attenuation of morphine-induced analgesia produced by these lesions was independent of alterations in regional NE content. DISCUSSION In accordance with previous reports47, 4s, microinjection of monosodium-Lglutamate (MSG) was an effective lesioning method. Gross examination of the injection sites revealed a slender cannula track which ended in a cylindrical or teardrop-shaped region of gliosis. The severe tissue disturbances which accompany electrolytic lesions were absent. Detailed examination of MSG injection sites 31 days after infusion disclosed a loss of cell bodies which was especially apparent when the lesion involved the densely populated LC. The loss of NE-containing cell bodies was confirmed by (1) the reduction in or lack of fluorescent catecholamine cell bodies in the LC, and (2) the reduction in NE content of the cortex, spinal cord, and medial brain stem, areas to which the LC projects. In this study, no attempt was made to determine the extent of damage to fiber tracts in the vicinity of the injection sites. It should be noted, however, that lesions of the PBV, an area that includes dense projections to and from the LC 49, failed to alter NE content of the three areas examined. This observation suggests that the glutamate lesions did not damage NE fibers passing through the injection site. More detailed studies have reported that axons of passage are not damaged by glutamate-induced lesions 2s,47,Sa. For example, light-microscopic examination of glutamate-induced lesions in the rostral hypothalamus revealed no more damage to axons of passage than that produced by insertion of the injection cannula alone 47. In addition, electron-microscopic examination of cortical sites following the iontophoretic application of glutamate revealed acute swelling of somata, dendrites, and axons in the area of the injection5s. However, these changes reversed within several days with the exception of neuronal perikarya which became electron-opaque and shrunken. Similarly, light-and electron-microscopic studies of sites in the rat striatum 21 days following glutamate injection indicated that all intrinsic striatal neurons had degenerated, but bundles of axons passing through the area of injection were intact and appeared normal 2s. Nociceptive thresholds determined using both the HP and TF tests were unaltered by lesions of the LC -k PBV or PBV alone when compared with the intact control group between 17 and 35 days after surgery. In contrast, increased nociceptive thresholds following electrolytic lesions of the LC have been reported by others using foot pinch43 and flinch jump s analgesiometric assays. However, these threshold alterations were temporary and both studies reported recovery of thresholds to near control values by approximately 20 days. Thus, in the present studies, lesion-induced changes in nociceptive threshold may have recovered by the time the animals were tested for the first time (17 days). In agreement with previous reports20, 43 lesions which involved the LC attenu-
88 ated morphine-induced analgesia. However, lesions which spared the LC, but involved the area lateral and ventral to it, the PBV, also produced attenuation of morphine analgesia. In fact, both lesions were equipotent in their capacity to obtund analgesia produced by systemic morphine. In the present study, bilateral lesions of the LC were found to include parts of the PBV situated ventral and lateral to the LC. Thus, the common factor responsible for the attenuation of morphine analgesia may be destruction of the PBV rather than the LC. In addition, examination of lesion sites described in previous studies 2°,43 supports the suggested involvement of the PBV in lesion-induced attenuation of morphine analgesia. Although the LC was damaged in each of these studies, the lesions also extended into the area ventral and lateral to the LC, the nucleus parabrachialis ventralis. The statistically significant reductions in cortical, spinal cord, and brain stem NE content following LC + PBV lesions confirms the destruction of NE-containing perikarya in the LC. This observation is consistent with the findings of other workersZ0, 43 and leads to the suggestion that destruction of NE-containing cells in the LC causes a reduction in the capacity of morphine to induce analgesia. However, if destruction of the NE-containing cells of the LC was responsible for the attenuation of analgesia, then alterations in the NE content of areas to which the LC projects would be expected to correlate with the lesion-induced effects on morphine. Such a relationship did not exist, since changes in cortical, cord, or medial brain stem NE content did not correlate with attenuation of morphine analgesia. Thus, destruction of the LC does not appear to alter morphine-induced analgesia. However, since attenuation of morphine effects was observed following PBV lesions which did not alter NE content in the three areas assayed, destruction of the PBV appears to be responsible for the lesion effects. This conclusion is in conflict with previous studies which attributed the attenuation of morphine analgesia to destruction of the LC and the attendant reduction in NE. However, the conclusion that destruction of NE-containing cells was responsible for reducing the effectiveness of morphine in these studies was based solely on an observed decrease in forebrain N E content. No attempt was made to correlate the reduction in NE with the decreased capacity of morphine to induce analgesia. If these two variables, NE content and the capacity of morphine to induce analgesia, are related, they would be expected to co-vary as a function of the extent to which the LC had been destroyed. For this reason, correlation of changes in morphine analgesia with changes in NE content provides a more accurate analysis of the involvement of NE in the mediation of lesion-induced changes in morphine analgesia than simply reporting average amine levels and average effects on morphine analgesia. These studies have demonstrated that (1) lesions which included both the LC and the PBV attenuated morphine-induced analgesia, but this attenuation did not correlate with alterations in NE content of three areas of the brain and (2) lesions restricted to the PBV alone which did not involve the LC attenuated morphine analgesia to a comparable extent. These observations lead to the conclusion that destruction of the LC is not responsible for the attenuation of morphine-induced analgesia. In addition, destruction of the area ventral and lateral to the LC, the PBV,
89 may be reponsible for the attenuation. Thus, the PBV may be involved in the expression of morphine analgesia. This suggestion is supported by reports demonstrating a high density of opiate receptors2 and a high concentration of the endogenous opioid ligand, enkephalin, localized in terminals41,46,51 and cell bodies in the PBV 15,16,5a. Furthermore, the PBV receives afferent projections from both the periaqueductal gray 38 and dorsal raphe nucleus7, 38 and in turn projects to these areas 39. Since numerous studies have demonstrated the involvement of these nuclei in the mediation of morphine analgesiaa4,25,56, the PBV may also be an integral part of the brain stem system which mediates opiate-induced analgesia. ACKNOWLEDGEMENTS
Supported by Grant NS 12649 from the U.S. Public Health Service. We thank Ms. Joann M. Hettasch and Ms. Linda J. Frederick for expert technical 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. J., Autoradiographic localization of opiate receptors in rat brain. II. The brain stem, Brain Research, 129 (1977) 1-12. 3 Ayhan, J. H., Effect of 6-hydroxydopamine on morphine analgesia, Psychopharmacol. (Berl.), 25 (1972) 183-188. 4 Bhargava, H. N., Afifi, A.H. and Way, E. L., Effect of chemical sympathectomy on morphine antinociception and tolerance development in the rat, Biochem. Pharmacol., 22 (1973) 2769-2772. 5 Bird, S. J. and Kuhar, M. J., Iontophoretic application of opiates to the locus coeruleus, Brain Research, 122 (1977) 523-533. 6 Blasig, J., Reinhold, K. and Herz, A., Effect of 6-hydroxydopamine, 5,6-dihydroxytryptamine and raphe lesions on the antinociceptive actions of morphine in rats, Psyehopharmacol. (BerL), 31 (1973) 111-119. 7 Bobillier, P., Segui, S., Petitjean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Research, 113 (1976), 449~[86. 8 Bodnar, R. J., Ackermann, R. F., Kelly, D. D. and Glusman, M., Elevations in nociceptive thresholds following locus coeruleus lesions, Brain Res. Bull., 3 (1978) 125-130. 9 Buxbaum, D. M., Yarbrough, G. G. and Carter, M. E., Biogenic amines and narcotic effects. I. Modification of morphine-induced analgesia and motor activity after alteration of cerebral amine levels, J. PharmaeoL exp. Ther., 185 (1973) 317-327. 10 Chang, C. C., A sensitive method for spectrophotofluorometric assay of catecholamines, J. Neuropharmacol., 3 (1964) 643-649. 11 Chu, N-S. and Bloom, F. E., The catecholamine-containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections, Brain Research, 66 (1974) 1-21. 12 De La Torte, J. C. and Surgeon, J. W., A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method, Histochemistry, 49 (1976) 81-93. 13 Fennessey, M. R. and Lee, J. R., Modification of morphine analgesia by drugs affecting adrenergic and tryptaminergic mechanisms, J. Pharm. Pharmacol., 22 (1970) 930-935. 14 Fields, H. L. and Basbaum, A. I., Brainstem control of spinal pain-transmission neurons, Ann. Rev. Physiol., 40 (1978) 217-248.
90 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 Johansson, O., Hokfelt, T., Elde, R. P., Schultzberg, M. and Terenius, L., Immunohistochemical distribution of enkephalin neurons. In E. Costa and M. Trabucchi (Eds.), Advances in Biochemical Psychopharmacology, Vol. 18, Raven Press, New York, 1978, pp. 51-70. 17 Keppel, G., Design and Analysis: A Researcher's Handbook, Prentice-Hall, New Jersey, 1973, 658 pp. 18 Kobayashi, R. M., Palkovits, M., Kopin, I. J. and Jacobowitz, D. M., Biochemical mapping of noradrenergic nerves arising from the rat locus coeruleus, Brain Research, 77 (1974) 269-279. 19 Korf, J., Bunney, B. S. and Aghajanian, G. K., Noradrenergic neurons: morphine inhibition of spontaneous activity, Europ. J. PharmacoL, 25 (1974) 165-169. 20 Kostowski, W., Jerlicz, M., Bidzinski, A. and Hauptmann, M., Reduced analgesic effects of morphine after bilateral lesions of the locus coeruleus in rats, Pol. J. PharmacoL Pharm., 30 (1978) 49-53. 21 Levy, R. A. and Proudfit, H. K., A comparison of the analgesic activity of baclofen with that of morphine following microinjection at sites in the rat brain stem, Europ. J. Pharmacol., 57 (1979) 43-65. 22 Lindvall, O. and Bjorklund, A., The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method, Acta physioL scand., Suppl. 412 (1974) 1-48. 23 Loizou, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain Research, 15 (1969) 563-566. 24 Major, C. T. and Pleuvry, B. J., Effects of a-methyl-p-tyrosine, p-chlorophenyl-alanine, L-/3(3, 4-dihydroxyphenyl)alanine, 5-hydroxytryptophan and diethyldithiocarbamate on the analgesic activity of morphine and methylamphetamine in the mouse, Brit. J. Pharmacol., 42 (1971) 512-521. 25 Mayer, D. J. and Price, D. D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379~04. 26 Montel, H., Starke, K. and Weber, F., Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 283 (1974) 357-369. 27 Nygren, L.G. and Olson, L., A new major projection from locus coeruleus: the main source of noradrenaline terminals in the ventral and dorsal columns of the spinal cord, Brain Research, 132 (1977) 85-93. 28 Olney, J. W. and DeGubareff, T., Glutamate neurotoxicity and Huntington's chorea, Nature (Lond.), 271 (1978), 557-559. 29 Olson, L. and FL'xe, K., On the projections from the locus coeruleus noradrenaline neurons: the cerebellar innervation, Brain Research, 28 (1971) 165-171. 30 Paalzow, G. and Paalzow, L., Morphine-induced inhibition of different pain responses in relation to the regional turnover of rat brain noradrenaline and dopamine, Psychopharmacol. (Berl.), 45 (1975) 9-20. 31 Pert, C. B., Kuhar, M. J. and Snyder, S. H., Autoradiographic localization of the opiate receptor in rat brain, Life Sci., 16 (1975) 1849-1854. 32 Pert, C. B., Kuhar, M. J. and Snyder, S. H., Opiate receptor: autoradiographic localization in rat brain, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 3729-3733. 33 Pickel, V. M., Segal, M. and Bloom F. E., A radioautographic study of the efferent pathways of the nucleus locus coeruleus, J. comp. NeuroL, 155 (1974) 15-42. 34 Price, M. T. and Fibiger, H. C., Ascending catecholamine systems and morphine analgesia, Brain Research, 99 (1975) 189-193. 35 Proudfit, H. K., Analgesia following microinjection of morphine into the nucleus raphe magnus of acutely decerebrate rats, Soc. NeuroscL, 3 (1977) 961. 36 Roizen, M. F. and Jacobowitz, D. M., Studies on the origin of innervation of the noradrenergic area bordering on the nucleus raphe dorsalis, Brain Research, 101 (1976) 561-568. 37 Ross, R. A. and Reis, D. J., Effects of lesions of locus coeruleus on regional distribution of dopamine-fl-hydroxylase activity in rat brain, Brain Research, 73 (1974) 161-166. 38 Sakai, K., Touret, M., Salvert, D., Leger, L. and Jouvet, M., Afferent projections to the cat locus coeruleus as visualized by the horseradish peroxidase technique, Brain Research, 119 (1977) 21-41. 39 Sakai, K., Salvert, D., Touret, M. and Jouvet, M., Afferent connections of the nucleus raphe
91
40 41 42 43
44
45 46
47
48 49
50 51
52 53 54
55 56
dorsalis in the cat as visualized by the horseradish peroxidase technique, Brain Research, 137 (1977) 11-35. Samanin, R. and Bernasconi, S., Effects of intraventricularly injected 6-OH dopamine or midbrain raphe lesion on morphine analgesia in rats, PsychopharmacoL (BerL), 25 (1972) 175-182. Sar, M., Stumpf, W. E., Miller, R. J., Chang, K-J. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. comp. NeuroL, 182 (1978) 17-38. Sasa, M., Munekiyo, K. and Takaori, S., Morphine interference with noradrenaline-mediated inhibition from the locus coeruleus, Life Sci., 17 (1975) 1373-1380. Sasa, M., Munekiyo, K., Osumi, Y. and Takaori, S., Attenuation of morphine analgesia in rats with lesions of the locus coeruleus and dorsal raphe nucleus, Europ. J. Pharmacol., 42 (1977) 53-62. Satoh, K., Tohyama, M., Yamamoto, K., Sakumoto, T. and Shimizu, N., Noradrenaline innervation of the spinal cord studied by the horseradish peroxidase method combined with monoamine oxidase staining, Brain Research, 30 (1977) 175-186. Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956, 312 pp. Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin: Immunohistochemical mapping in rat central nervous system, Proc. nat. Acad. Sci. (Wash.), 75 (1977) 2167-2171. Simson, E. L., Gold, R. M., Standish, L. J. and Pellett, P. L., Axon-sparing brain lesioning technique: the use of monosodium-L-glutamate and other amino acids, Science, 198 (1977) 515-517. Simson, E. L. and Gold, R. M., Axon-sparing brain lesioning technique, Science, 200 (1978) 1417. Swanson, L. W. and Hartman, B. K., The central adrenergic system. An immuno-fluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopaminefl-hydroxylase as a marker, J. comp. NeuroL, 163 (1975) 467-506. Tohyama, M., Maeda, T. and Shimizu, N., Detailed noradrenaline pathways of locus coeruleus neuron to the cerebral cortex with use of 6-hydroxydopa, Brain Research, 79 (1974) 139-144. Uhl, G. R., Goodman, R. R., Kuhar, M. J., Childers, S. R. and Snyder, S. H., Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Research, 166 (1979) 75-94. Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physioL scand., 82, Suppl. 367 (1971) 148. Van Harreveld, A. and Fifkova, E., Light- and electron-microscopic changes in central nervous tissue after electrophoretic injection of glutamate, Exp. Molec. Path., 15 (1971) 61-81. Welch, A. S. and Welch, B. L., Solvent extraction method for simultaneous determination of norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid in a single mouse brain, Analyt. Biochem., 30 (1969) 161-179. Yaksh, T. L. and Rudy, T. A., Studies on the direct spinal action of narcotics in the production of analgesia in the rat, J. Pharrnacol. exp. Ther., 202 (1977) 411428. Yaksh, T. L. and Rudy, T. A., Narcotic analgetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359.
79
EFFECTS OF LOCUS COERULEUS LESIONS ON MORPHINE-INDUCED ANTINOCICEPTION
DONNA L. HAMMOND and HERBERTK. PROUDFIT University of Illinois, Department of Pharmacology, College of Medicine, Chicago, Ill. 60680 (U. S..4.)
(Accepted June 9th, 1979) Key words: antinociception -- locus coeruleus -- morphine -- norepinephrine -- ventral para-
brachial nucleus
SUMMARY These studies were designed to examine the role of the norepinephrine-containing cells comprising the nucleus locus coeruleus (LC) in the mediation of pain perception and morphine-induced antinociception. Nociceptive threshold and morphine-induced analgesia were measured following monosodium-L-glutamate lesions of the LC and adjacent tegmentum (nucleus parabrachialis ventralis; PBV) at 17, 24 and 31 days after surgery. Nociceptive thresholds assessed by the tail flick and hot plate assays were not altered following lesions which included both the LC and PBV (Group l) or by lesions of the PBV (Group 2) alone. Examination of lesion-induced effects on the capacity of morphine to induce analgesia revealed that damage which included both LC and PBV as well as that confined primarily to the PBV resulted in attenuation of analgesia induced by morphine. Those lesions which involved the LC altered norepinephrine content in the cortex, spinal cord and medial brain stem; however, no correlation could be demonstrated between the attenuation of morphine-induced analgesia and the changes in norepinephrine content of the brain regions examined. Thus, destruction of the LC does not appear to be responsible for the decreased effectiveness of morphine. The only region consistently damaged in both groups 1 and 2 was the ventral parabrachial nucleus. Therefore, we tentatively conclude that destruction of the PBV was responsible for the observed attenuation of morphine analgesia.
INTRODUCTION The participation of brain norepinephrine (NE) systems in the mediation of morphine analgesia has been suggested by several lines of pharmacological evidence. However, the precise role of NE is obscured by the contradictory results of these
80 studies. For example, depletion of whole brain N E by various agents has been reported to antagonize~,4,6, '~4, potentiateg,~o, 40 or exert no effect 13 on morphineinduced analgesia. These contradictory results may in part be accounted for by differences in species, analgesiometric tests, doses and pretreatment schedules utilized. However, it is also likely that complex mechanisms are involved in the relationship between NE systems and those mediating morphine analgesia; some perhaps facilitating and others opposing the actions of morphine. Thus, studies of discrete NEcontaining nuclei might be expected to yield less equivocal results than those using pharmacological agents which produce general and indiscriminant alterations in whole brain NE levels. One such NE-containing nucleus, the nucleus locus coeruleus (LC), has been suggested to be involved in the expression of morphine-induced analgesia. For example, cells in the LC respond to noxious peripheral stimulation with a marked, though transient, increase in firing 19. In addition, the systemiO ,~ or iontophoretic 5 application of morphine to these cells produces a naloxone-reversible depression of firing. The sensitivity of the LC neurons to morphine is not unexpected in view of the high density of opiate receptors it possesses 2,31,3z. Morphine has also been suggested to inhibit the release of NE from axon terminals originating in the LCa2; a similar action of morphine has been demonstrated in cortical slices 26. A role for the LC in the actions of opiates has also been suggested by anatomical studies demonstrating that the LC is the origin of a diffuse network of projections to the cerebral, hippocampal and cerebellar corticeslS,'~2, 29,a:3,37,5°,52 in addition to various subcortical areaslS,"2,2a,aT, '~2. A salient feature of these projections is their presence in areas known to be involved in the mediation of morphine analgesia such as the periaqueductal gray z3,a7, n. raphe dorsalis 23,36 and n. raphe magnus it. Recently, a direct coeruleo-spinal projection has been described ~7,4~, a p~rtion of which terminates in the superficial layers of the dorsal horn. Furthermore, lesions of the LC and its projections have been shown to alter morphine-induced analgesia. For example, 6-hydroxydopamine-induced lesions of the dorsal noradrenergic bundle (DNB) have been reported to potentiate morphine analgesia, although destruction of neither the dorsal nor the ventral bundle altered sensitivity to pain 34. Since the LC has been identified as the principal source of fibers ill the DNB 22,5°,52, these data suggest that the LC may have an inhibitory influence on systems that mediate morphine-induced analgesia. However, other studies using electrolytic lesions of the LC have reported increases in nociceptive threshold 8,43 and attenuation of morphine analgesia "°,~3 assessed by flinch jump and compression tests. The interpretation of these latter findings is somewhat difficult since electrolytic lesions damage axons of passage and blood vessels in addition to cell perikarya in the target area. Thus, damage to adjacent fiber tracts may contribute to the reported effect of LC lesions and may account for the different results obtained using electrolytic and 6OH DA-induced lesions. The present studies were designed to circumvent these problems by investigating the effects of LC destruction on nociceptive threshold and morphine-induced analgesia using lesions produced by the local injection of monosodium-L-glutamate. This
81 method has been reported to spare axons of passage, blood vessels and glia while producing destruction of cell perikarya restricted to the area of injection 28,47,48,53. In addition, previous studies only measured changes in forebrain NE levels following LC lesions. However, the LC also projects to the medial brain stem 11 and spinal cord dorsal horn 27,44, sites at which the local application of morphine has been shown to induce analgesia1,21,35, ~5. For this reason, we examined the effect of LC lesions on N E content in the spinal cord, cortex and the medial brain stem in an attempt to ascertain the existence of correlations between lesion-induced changes in NE levels in these areas and changes in morphine-induced analgesia. METHODS
Lesioning procedure Female Sprague-Dawley-derived rats weighing between 220 and 280 g were used for these studies. The animals were anesthetized with sodium pentobarbital, placed in a stereotaxic frame, and the tip of a 28-gauge stainless steel injection cannula was stereotaxically positioned in the locus coeruleus. The injection cannula was inclined at a 20 ° angle to avoid rupturing the superior sagittal sinus. The injection system consisted of a 40 cm length of polyethylene (PE-20) tubing connecting the injection cannula with a 5/zl Hamilton syringe which was driven by an infusion pump. Bilateral lesions were made by injecting 5 #1 of 1 ~ monosodium-L-glutamate in saline at a rate of 0.59 /A/rain into each of 4 sites within the LC (two injections per side). The stereotaxic coordinates for these injection sites were as follows: P 1.6, H 1.8, L ± 1.3 m m and P 1.2, H 2.0, L --4- 1.3 m m with the incisor bar at + 5 . 0 mm. The injection of the glutamate solution was monitored by observing the movement of an air bubble in the tubing. The cannula was left in place for an additional 2 min to minimize back diffusion when it was withdrawn. The animals were housed in groups of 5 and allowed 17 days to recover from the effects of surgery.
Analgesiometric tests Two analgesiometric tests (tail flick and hot plate) were utilized to acertain alterations in nociceptive threshold and morphine analgesia. Tail flick latency (TFL) was defined as the elapsed time between onset of a high intensity light beam focused on the blackened tail and the reflex tail flick response. The average of three determinations was recorded. The number of seconds which elapsed before the rat licked its hind paw or jumped after placement on a 55 °C hot plate was defined as the hot plate latency (HPL). To avoid injury, cut-offvalues of 14 and 40 sec were chosen for the tail flick and hot plate, respectively. T F L and H P L were expressed as an antinociceptive index (AI) to minimize variability due to differences in pre-drug latencies and thus allow more valid comparisons among animals. The AI was calculated as follows: AI
=
latency after morphine - - control latency , , . An/-xl of 1.0 denotes antinociception cut-off latency - - control latency
of a magnitude equal to or greater than the appropriate cut-off latency, while an AI of 0.0 denotes absence of antinociception. Negative values indicate hyperalgesia.
82
Assessment of lesion effects Nociceptive threshold. Prior to surgery, tail flick and hot plate latencies were determined and these values were compared with those obtained 17, 24, and 31 days following surgery. Non-lesioned control animals were tested concurrently. The rats were first tested on the tail flick apparatus and then immediately tested on the hot plate. Morphine-induced analgesia. Following assessment of the nociceptive threshold on each of the three test days, the capacity of systemic morphine to induce analgesia in both lesioned and non-lesioned control animals was examined. Each group of rats received doses of 2.0, 5.0, and 10.0 mg/kg morphine sulfate (Merck) subcutaneously on days 17, 24, and 31, respectively. Drug effects were assessed 30 min following injection, the time previously determined as the peak of morphine-induced analgesia.
NE determination NE content in the cortex, lumbosacral spinal cord, and medial brain stem was determined spectrofluorometrically using a modification of the method reported by Welch and Welch 54 for extraction of the amine and the method of Chang 1° for development of the fluorophore. Following decapitation, the spinal cord was rapidly removed, all roots were dissected away and that portion of the cord caudal to and including the lumbar enlargement was assayed. The brain was quickly removed from the calvarium and a slice of cortex was dissected free of the underlying diencephalon. The brain stem was then blocked and a 2 mm thick parasagittal segment containing the raphe complex removed for assay. The regional NE content of lesioned animals was expressed as a percentage of NE levels in corresponding areas of non-lesioned animals.
Histology Histological verification of LC destruction following glutamate lesions was made in all lesioned animals. The degree of damage sustained by the LC in lesioned animals was determined by comparison with brain sections from intact control animals in which the distribution of NE-containing cells in the LC had been determined by fluorescence histochemical visualization. Identification of the lesion site in l 1 animals was based on the examination of 40 # m coronal sections taken every 200 #m and stained with cresyl violet only. In the remaining 9 animals, LC damage was determined using fluorescence histochemical methods in addition to routine histological examination. The brains from these animals were removed, the appropriate block of tissue frozen and l0 #m sections taken at 200 #m intervals through the LC. The sections were reacted with glyoxylic acid 12 to produce a NE fluorophore and examined using fluorescence microscopy. Alternate 30 #m sections of these brains were stained with cresyl violet.
Statistical analysis Post-lesion TFLs and HPLs were expressed as a percentage of pre-lesion values. Post-lesion nociceptive thresholds of lesioned animals were compared with those of
83
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Fig. 1. Camera lucida drawings illustrating the location of representative monosodium-L-glutamateinduced lesions in the dorsolateral pontine tegmentum. A: intact control; B : lesion including thelocus coeruleus and the ventral parabrachial nucleus (LC + PBV); C: lesion restricted primarily to the ventral parabrachial nucleus (PBV). Abbreviations: CER, cerebellum; el, ependymal lining; IV, fourth ventricle; LC, nucleus locus coeruleus; MeV, mesencephalic nucleus of the trigeminal; PBV, nucleus parabrachialis ventralis; SCP, superior cerebellar peduncle. intact control animals with a two-way analysis of variance with replications and N e w m a n - K e u l s test for multiple comparisons 17. The response of lesioned animals to various doses of morphine was compared to that of non-lesioned control animals by means of a two-way analysis of variance with replications and N e w m a n - K e u l s test. Spearman's Rank Correlation Coefficient 45 was utilized to assess the degree of association between regional changes in N E content and alterations in morphineinduced analgesia. RESULTS Lesion site
Glutamate-induced lesions examined in sections stained with cresyl violet appeared as cylindrical or teardrop-shaped regions of glialproliferation exhibiting marked loss of neuronal perikarya. The size of the lesion at the site of injection was
84 usually between 0.3 mm and 0.6 mm in diameter. The glutamate solution apparently spread dorsally along the cannula resulting in an elongated pattern of gliosis (Fig. 1B and C). No sign of physical damage due to the large injection volume was seen; the track produced by insertion of the injection cannula was the only observable evidence of destruction by physical forces. Fluorescence histochemical observations of sections taken through the LC revealed a reduction and in many cases a complete absence of catecholamine fluorescence with lesions localized to the LC. A given animal was considered to have received a lesion of the LC if the following criteria were met: (1) the presence o f a cannula track in the LC, (2) loss of cell bodies and increased gliosis in the LC, (3) reduction or lack of catecholamine fluorescence in the LC, and (4) reduction of cortical NE to less than 80 ~ of control values. Accordingly, 8 animals possessed bilateral lesions of the LC. In 7 of the 8 animals with bilateral LC lesions, the cannula track, gliosis, and loss of cell bodies also extended beneath the LC to the nucleus parabrachialis ventralis (PBV). Such a lesion is illustrated in Fig. 1B. The lesions in these 7 animals were considered to occupy similar sites and will be referred to as LC 4- PBV-lesioned animals. In the remaining bilaterally lesioned animal, damage was restricted to the LC only. Three animals had unilateral lesions of the LC; however, due to technical difficulties the precise extent of the lesion could not be determined. These animals were not included in any group for evaluation of lesion-induced alterations in the nociceptive threshold or morphineinduced analgesia. However, they were used to assess the relationship between alterations in regional NE content and alterations in morphine-induced analgesia. Nine animals did not have lesions of the LC. In 7 of these animals the cannula track and gliosis appeared lateral to the LC and extended ventrally to include the PBV, either unilaterally (n = 6) or bilaterally (n -- 1). Such a lesion is illustrated in Fig. 1C. These animals were grouped together and will be referred to as PBV-lesioned animals. The two remaining animals were not included in any group due to difficulty in defining the precise extent of the lesions. These animals were used to assess the relationship between alterations in NE content and alterations in morphine-induced analgesia. Regional N E content Bilateral lesions of the LC significantly reduced cortical, cord, and medial brain stem NE content to 53.03 4- 4.99~/o (n = 6), 72.82 J_ 4.80~ (n 7), and 86.74 ± 2.88 ~ (n = 3) of control (mean ± S.E.M.), respectively (Student t-test, P < 0.05, all cases). Absolute levels of NE expressed as ng/g wet weight tissue (mean ~ S.E.M.) in corresponding areas of non-lesioned control animals were 246.48 ~_ 17.35 (n = 8), 269.49 ~ 16.85 (n = 9), and 604.70 ~ 24.84 (n 4) for cortex, spinal cord and medial brain stem, respectively. Followinglesions which produced varying degrees of damage to the LC, NE levels ranged from 79.29 to 181.70 ng/g in cortex; 152.63 to 330.0 ng/g in the spinal cord; and 492.31 to 551.99 ng/g in the medial brain stem. Lesions of the PBV which spared the LC did not alter the regional NE content significantly. In these animals, cortical, cord and medial brain stem NE levels were 93.01 A 4.29)o (n ~ 7), 85.30 ~ 9.50 °/o (n = 7), and 98.21 ~ 7.98 ~ (n = 3) of control respectively (Student ttest, P ~ 0.05, all cases).
85
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Fig. 2. The effect of lesions on the dose-response relationship of morphine administered to nonlesioned control rats (0 n = 19), LC + PBV-lesioned rats (ll n = 7), and PBV-lesioned rats (A n = 7). The antinociceptive index is plotted on the ordinate and the log dose is plotted on the abscissa. Each point represents the mean AI 4- S.E.M.
Behavioral effects of lesions None of the glutamate-lesioned animals in this study exhibited any behavioral alterations or motor deficits. Animals were active throughout the duration of the experiment and exhibited normal exploratory behavior. All animals displayed normal feeding and drinking patterns accompanied by normal weight gain. These animals were essentially indistinguishable from unoperated controls.
Alterations in nocieeptive threshold The capacity of the various lesions to produce alterations in reactivity to noxious stimuli was assessed using both the tail flick and hot plate tests. The statistical significance of alterations in nociceptive threshold was determined using a two-way analysis of variance 17 which compared response latencies of the three groups of animals (LC 6- PBV, PBV, and intact control) measured at 17, 24, and 31 days following surgery. This analysis revealed no statistically significant alterations in nociceptive threshold for any of the three groups as a function of time after surgery and no differences between the intact control group and either of the lesion groups compared at the three time points.
Alterations in morphine-induced analgesia Fig. 2 illustrates the capacity of various doses of morphine to elevate the T F L and H P L in intact control, LC ÷ PBV, and PBV-lesioned animals. These data indicate that lesions of both the LC 6- PBV and the PBV resulted in potent attenuation of morphine-induced analgesia regardless of the analgesiometric test. Both the lesioned and non-lesioned control animals responded to morphine in a statistically significant dose-related fashion (two-way ANOVA, P < 0.05). However, the magnitude of the
86 Spinal
Cord
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oc
.00 Mediol 8roinstern
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Fig. 3. Correlation between the magnitude of antinociception (AI) induced by morphine sulfate (5 mg/kg) and NE levels in (A) spinal cord, (B) cortex, and (C) medial brain stem. Ordinate: antinociceptive index (AI) derived from tail flick latency measurements. Abscissa : NE levels in the spinal cord, cortex, and medial brain stem determined in each animal and expressed as per cent of NE in corresponding areas of intact control animals. Spearman's Rank Correlation Coefficients for spinal cord = -0.01 ; cortex = 0.09; medial brain stem = 0.04. analgesia produced by identical doses of morphine administered to both the LC + PBV and PBV-lesioned animals was significantly less than that produced in nonlesioned control animals as assessed by either HP or TF tests (Newman-Keuls, P < 0.05). Furthermore, the log dose-response curve of the LC ÷ PBV-lesioned animals was not significantly different from that of the PBV-lesioned animals ascertained by either analgesiometric test (Newman-Keuls, P > 0.05). Thus, both lesion placements were equipotent in their ability to attenuate morphine-induced analgesia.
Regional NE content and morphine analgesia The relationship between alterations in the capacity of morphine to elevate the TFL and HPL and alterations in regional NE content following lesions of the LC + PBV and PBV was also examined. Fig. 3 illustrates the correlation between NE content of the spinal cord, cortex, and medial brain stem and analgesia induced by administration of morphine sulfate (5 mg/kg) to lesioned animals using the tail flick test. Assessment of the degree of association between changes in cortical, spinal cord, or medial brain stem NE levels and the response of lesioned animals to morphine was made using Spearman's Rank Correlation CoefficienOL No significant correlation existed between these two variables for any of the brain regions examined (rs = --0.01, 0.09, and 0.04, respectively; P > 0.05). Similar results were obtained using the hot plate test. Spearman's Rank Correlation Coefficients for these data were 0.02, ~ ) . 3 3 , and 0.08 for cortical, spinal cord, and brain stem NE content versus morphine
87 analgesia, respectively. None of these coefficients were statistically significant (P > 0.05). Furthermore, no significant correlations were observed between the analgesia resulting from either 2 or l0 mg/kg of morphine and NE content in any of the areas examined. Thus, the attenuation of morphine-induced analgesia produced by these lesions was independent of alterations in regional NE content. DISCUSSION In accordance with previous reports47, 4s, microinjection of monosodium-Lglutamate (MSG) was an effective lesioning method. Gross examination of the injection sites revealed a slender cannula track which ended in a cylindrical or teardrop-shaped region of gliosis. The severe tissue disturbances which accompany electrolytic lesions were absent. Detailed examination of MSG injection sites 31 days after infusion disclosed a loss of cell bodies which was especially apparent when the lesion involved the densely populated LC. The loss of NE-containing cell bodies was confirmed by (1) the reduction in or lack of fluorescent catecholamine cell bodies in the LC, and (2) the reduction in NE content of the cortex, spinal cord, and medial brain stem, areas to which the LC projects. In this study, no attempt was made to determine the extent of damage to fiber tracts in the vicinity of the injection sites. It should be noted, however, that lesions of the PBV, an area that includes dense projections to and from the LC 49, failed to alter NE content of the three areas examined. This observation suggests that the glutamate lesions did not damage NE fibers passing through the injection site. More detailed studies have reported that axons of passage are not damaged by glutamate-induced lesions 2s,47,Sa. For example, light-microscopic examination of glutamate-induced lesions in the rostral hypothalamus revealed no more damage to axons of passage than that produced by insertion of the injection cannula alone 47. In addition, electron-microscopic examination of cortical sites following the iontophoretic application of glutamate revealed acute swelling of somata, dendrites, and axons in the area of the injection5s. However, these changes reversed within several days with the exception of neuronal perikarya which became electron-opaque and shrunken. Similarly, light-and electron-microscopic studies of sites in the rat striatum 21 days following glutamate injection indicated that all intrinsic striatal neurons had degenerated, but bundles of axons passing through the area of injection were intact and appeared normal 2s. Nociceptive thresholds determined using both the HP and TF tests were unaltered by lesions of the LC -k PBV or PBV alone when compared with the intact control group between 17 and 35 days after surgery. In contrast, increased nociceptive thresholds following electrolytic lesions of the LC have been reported by others using foot pinch43 and flinch jump s analgesiometric assays. However, these threshold alterations were temporary and both studies reported recovery of thresholds to near control values by approximately 20 days. Thus, in the present studies, lesion-induced changes in nociceptive threshold may have recovered by the time the animals were tested for the first time (17 days). In agreement with previous reports20, 43 lesions which involved the LC attenu-
88 ated morphine-induced analgesia. However, lesions which spared the LC, but involved the area lateral and ventral to it, the PBV, also produced attenuation of morphine analgesia. In fact, both lesions were equipotent in their capacity to obtund analgesia produced by systemic morphine. In the present study, bilateral lesions of the LC were found to include parts of the PBV situated ventral and lateral to the LC. Thus, the common factor responsible for the attenuation of morphine analgesia may be destruction of the PBV rather than the LC. In addition, examination of lesion sites described in previous studies 2°,43 supports the suggested involvement of the PBV in lesion-induced attenuation of morphine analgesia. Although the LC was damaged in each of these studies, the lesions also extended into the area ventral and lateral to the LC, the nucleus parabrachialis ventralis. The statistically significant reductions in cortical, spinal cord, and brain stem NE content following LC + PBV lesions confirms the destruction of NE-containing perikarya in the LC. This observation is consistent with the findings of other workersZ0, 43 and leads to the suggestion that destruction of NE-containing cells in the LC causes a reduction in the capacity of morphine to induce analgesia. However, if destruction of the NE-containing cells of the LC was responsible for the attenuation of analgesia, then alterations in the NE content of areas to which the LC projects would be expected to correlate with the lesion-induced effects on morphine. Such a relationship did not exist, since changes in cortical, cord, or medial brain stem NE content did not correlate with attenuation of morphine analgesia. Thus, destruction of the LC does not appear to alter morphine-induced analgesia. However, since attenuation of morphine effects was observed following PBV lesions which did not alter NE content in the three areas assayed, destruction of the PBV appears to be responsible for the lesion effects. This conclusion is in conflict with previous studies which attributed the attenuation of morphine analgesia to destruction of the LC and the attendant reduction in NE. However, the conclusion that destruction of NE-containing cells was responsible for reducing the effectiveness of morphine in these studies was based solely on an observed decrease in forebrain N E content. No attempt was made to correlate the reduction in NE with the decreased capacity of morphine to induce analgesia. If these two variables, NE content and the capacity of morphine to induce analgesia, are related, they would be expected to co-vary as a function of the extent to which the LC had been destroyed. For this reason, correlation of changes in morphine analgesia with changes in NE content provides a more accurate analysis of the involvement of NE in the mediation of lesion-induced changes in morphine analgesia than simply reporting average amine levels and average effects on morphine analgesia. These studies have demonstrated that (1) lesions which included both the LC and the PBV attenuated morphine-induced analgesia, but this attenuation did not correlate with alterations in NE content of three areas of the brain and (2) lesions restricted to the PBV alone which did not involve the LC attenuated morphine analgesia to a comparable extent. These observations lead to the conclusion that destruction of the LC is not responsible for the attenuation of morphine-induced analgesia. In addition, destruction of the area ventral and lateral to the LC, the PBV,
89 may be reponsible for the attenuation. Thus, the PBV may be involved in the expression of morphine analgesia. This suggestion is supported by reports demonstrating a high density of opiate receptors2 and a high concentration of the endogenous opioid ligand, enkephalin, localized in terminals41,46,51 and cell bodies in the PBV 15,16,5a. Furthermore, the PBV receives afferent projections from both the periaqueductal gray 38 and dorsal raphe nucleus7, 38 and in turn projects to these areas 39. Since numerous studies have demonstrated the involvement of these nuclei in the mediation of morphine analgesiaa4,25,56, the PBV may also be an integral part of the brain stem system which mediates opiate-induced analgesia. ACKNOWLEDGEMENTS
Supported by Grant NS 12649 from the U.S. Public Health Service. We thank Ms. Joann M. Hettasch and Ms. Linda J. Frederick for expert technical 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. J., Autoradiographic localization of opiate receptors in rat brain. II. The brain stem, Brain Research, 129 (1977) 1-12. 3 Ayhan, J. H., Effect of 6-hydroxydopamine on morphine analgesia, Psychopharmacol. (Berl.), 25 (1972) 183-188. 4 Bhargava, H. N., Afifi, A.H. and Way, E. L., Effect of chemical sympathectomy on morphine antinociception and tolerance development in the rat, Biochem. Pharmacol., 22 (1973) 2769-2772. 5 Bird, S. J. and Kuhar, M. J., Iontophoretic application of opiates to the locus coeruleus, Brain Research, 122 (1977) 523-533. 6 Blasig, J., Reinhold, K. and Herz, A., Effect of 6-hydroxydopamine, 5,6-dihydroxytryptamine and raphe lesions on the antinociceptive actions of morphine in rats, Psyehopharmacol. (BerL), 31 (1973) 111-119. 7 Bobillier, P., Segui, S., Petitjean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Research, 113 (1976), 449~[86. 8 Bodnar, R. J., Ackermann, R. F., Kelly, D. D. and Glusman, M., Elevations in nociceptive thresholds following locus coeruleus lesions, Brain Res. Bull., 3 (1978) 125-130. 9 Buxbaum, D. M., Yarbrough, G. G. and Carter, M. E., Biogenic amines and narcotic effects. I. Modification of morphine-induced analgesia and motor activity after alteration of cerebral amine levels, J. PharmaeoL exp. Ther., 185 (1973) 317-327. 10 Chang, C. C., A sensitive method for spectrophotofluorometric assay of catecholamines, J. Neuropharmacol., 3 (1964) 643-649. 11 Chu, N-S. and Bloom, F. E., The catecholamine-containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections, Brain Research, 66 (1974) 1-21. 12 De La Torte, J. C. and Surgeon, J. W., A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method, Histochemistry, 49 (1976) 81-93. 13 Fennessey, M. R. and Lee, J. R., Modification of morphine analgesia by drugs affecting adrenergic and tryptaminergic mechanisms, J. Pharm. Pharmacol., 22 (1970) 930-935. 14 Fields, H. L. and Basbaum, A. I., Brainstem control of spinal pain-transmission neurons, Ann. Rev. Physiol., 40 (1978) 217-248.
90 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 Johansson, O., Hokfelt, T., Elde, R. P., Schultzberg, M. and Terenius, L., Immunohistochemical distribution of enkephalin neurons. In E. Costa and M. Trabucchi (Eds.), Advances in Biochemical Psychopharmacology, Vol. 18, Raven Press, New York, 1978, pp. 51-70. 17 Keppel, G., Design and Analysis: A Researcher's Handbook, Prentice-Hall, New Jersey, 1973, 658 pp. 18 Kobayashi, R. M., Palkovits, M., Kopin, I. J. and Jacobowitz, D. M., Biochemical mapping of noradrenergic nerves arising from the rat locus coeruleus, Brain Research, 77 (1974) 269-279. 19 Korf, J., Bunney, B. S. and Aghajanian, G. K., Noradrenergic neurons: morphine inhibition of spontaneous activity, Europ. J. PharmacoL, 25 (1974) 165-169. 20 Kostowski, W., Jerlicz, M., Bidzinski, A. and Hauptmann, M., Reduced analgesic effects of morphine after bilateral lesions of the locus coeruleus in rats, Pol. J. PharmacoL Pharm., 30 (1978) 49-53. 21 Levy, R. A. and Proudfit, H. K., A comparison of the analgesic activity of baclofen with that of morphine following microinjection at sites in the rat brain stem, Europ. J. Pharmacol., 57 (1979) 43-65. 22 Lindvall, O. and Bjorklund, A., The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method, Acta physioL scand., Suppl. 412 (1974) 1-48. 23 Loizou, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain Research, 15 (1969) 563-566. 24 Major, C. T. and Pleuvry, B. J., Effects of a-methyl-p-tyrosine, p-chlorophenyl-alanine, L-/3(3, 4-dihydroxyphenyl)alanine, 5-hydroxytryptophan and diethyldithiocarbamate on the analgesic activity of morphine and methylamphetamine in the mouse, Brit. J. Pharmacol., 42 (1971) 512-521. 25 Mayer, D. J. and Price, D. D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379~04. 26 Montel, H., Starke, K. and Weber, F., Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 283 (1974) 357-369. 27 Nygren, L.G. and Olson, L., A new major projection from locus coeruleus: the main source of noradrenaline terminals in the ventral and dorsal columns of the spinal cord, Brain Research, 132 (1977) 85-93. 28 Olney, J. W. and DeGubareff, T., Glutamate neurotoxicity and Huntington's chorea, Nature (Lond.), 271 (1978), 557-559. 29 Olson, L. and FL'xe, K., On the projections from the locus coeruleus noradrenaline neurons: the cerebellar innervation, Brain Research, 28 (1971) 165-171. 30 Paalzow, G. and Paalzow, L., Morphine-induced inhibition of different pain responses in relation to the regional turnover of rat brain noradrenaline and dopamine, Psychopharmacol. (Berl.), 45 (1975) 9-20. 31 Pert, C. B., Kuhar, M. J. and Snyder, S. H., Autoradiographic localization of the opiate receptor in rat brain, Life Sci., 16 (1975) 1849-1854. 32 Pert, C. B., Kuhar, M. J. and Snyder, S. H., Opiate receptor: autoradiographic localization in rat brain, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 3729-3733. 33 Pickel, V. M., Segal, M. and Bloom F. E., A radioautographic study of the efferent pathways of the nucleus locus coeruleus, J. comp. NeuroL, 155 (1974) 15-42. 34 Price, M. T. and Fibiger, H. C., Ascending catecholamine systems and morphine analgesia, Brain Research, 99 (1975) 189-193. 35 Proudfit, H. K., Analgesia following microinjection of morphine into the nucleus raphe magnus of acutely decerebrate rats, Soc. NeuroscL, 3 (1977) 961. 36 Roizen, M. F. and Jacobowitz, D. M., Studies on the origin of innervation of the noradrenergic area bordering on the nucleus raphe dorsalis, Brain Research, 101 (1976) 561-568. 37 Ross, R. A. and Reis, D. J., Effects of lesions of locus coeruleus on regional distribution of dopamine-fl-hydroxylase activity in rat brain, Brain Research, 73 (1974) 161-166. 38 Sakai, K., Touret, M., Salvert, D., Leger, L. and Jouvet, M., Afferent projections to the cat locus coeruleus as visualized by the horseradish peroxidase technique, Brain Research, 119 (1977) 21-41. 39 Sakai, K., Salvert, D., Touret, M. and Jouvet, M., Afferent connections of the nucleus raphe
91
40 41 42 43
44
45 46
47
48 49
50 51
52 53 54
55 56
dorsalis in the cat as visualized by the horseradish peroxidase technique, Brain Research, 137 (1977) 11-35. Samanin, R. and Bernasconi, S., Effects of intraventricularly injected 6-OH dopamine or midbrain raphe lesion on morphine analgesia in rats, PsychopharmacoL (BerL), 25 (1972) 175-182. Sar, M., Stumpf, W. E., Miller, R. J., Chang, K-J. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. comp. NeuroL, 182 (1978) 17-38. Sasa, M., Munekiyo, K. and Takaori, S., Morphine interference with noradrenaline-mediated inhibition from the locus coeruleus, Life Sci., 17 (1975) 1373-1380. Sasa, M., Munekiyo, K., Osumi, Y. and Takaori, S., Attenuation of morphine analgesia in rats with lesions of the locus coeruleus and dorsal raphe nucleus, Europ. J. Pharmacol., 42 (1977) 53-62. Satoh, K., Tohyama, M., Yamamoto, K., Sakumoto, T. and Shimizu, N., Noradrenaline innervation of the spinal cord studied by the horseradish peroxidase method combined with monoamine oxidase staining, Brain Research, 30 (1977) 175-186. Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956, 312 pp. Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin: Immunohistochemical mapping in rat central nervous system, Proc. nat. Acad. Sci. (Wash.), 75 (1977) 2167-2171. Simson, E. L., Gold, R. M., Standish, L. J. and Pellett, P. L., Axon-sparing brain lesioning technique: the use of monosodium-L-glutamate and other amino acids, Science, 198 (1977) 515-517. Simson, E. L. and Gold, R. M., Axon-sparing brain lesioning technique, Science, 200 (1978) 1417. Swanson, L. W. and Hartman, B. K., The central adrenergic system. An immuno-fluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopaminefl-hydroxylase as a marker, J. comp. NeuroL, 163 (1975) 467-506. Tohyama, M., Maeda, T. and Shimizu, N., Detailed noradrenaline pathways of locus coeruleus neuron to the cerebral cortex with use of 6-hydroxydopa, Brain Research, 79 (1974) 139-144. Uhl, G. R., Goodman, R. R., Kuhar, M. J., Childers, S. R. and Snyder, S. H., Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Research, 166 (1979) 75-94. Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physioL scand., 82, Suppl. 367 (1971) 148. Van Harreveld, A. and Fifkova, E., Light- and electron-microscopic changes in central nervous tissue after electrophoretic injection of glutamate, Exp. Molec. Path., 15 (1971) 61-81. Welch, A. S. and Welch, B. L., Solvent extraction method for simultaneous determination of norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid in a single mouse brain, Analyt. Biochem., 30 (1969) 161-179. Yaksh, T. L. and Rudy, T. A., Studies on the direct spinal action of narcotics in the production of analgesia in the rat, J. Pharrnacol. exp. Ther., 202 (1977) 411428. Yaksh, T. L. and Rudy, T. A., Narcotic analgetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359.