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N. raphe magnus lesions disrupt stimulation-produced analgesia from ventral but not dorsal midbrain areas in the rat
Brain Research, 261 (1983) 53-57 Elsevier Biomedical Press
53
N. Raphe Magnus Lesions Disrupt Stimulation-Produced Analgesia from Ventral but not Dorsal Midbrain Areas in the Rat G. J. PRIETO*, J. T. CANNON** and J. C. LIEBESKIND*** Department of Psychology, UCLA, Los Angeles, CA 90024, (U.S.A.)
(Accepted July 20th, 1982) Key words: stimulation-produced analgesia - - pain - - analgesia - - n. raphe magnus - periaqueductal gray matter - - opioid peptides
We previously found that the opiate antagonist, naloxone, partially blocks stimulation-produced analgesia (SPA) elicited from ventral but not dorsal regions of the medial midbrain in rats. The present study compares the effects of n. raphe magnus (NRM) lesions on SPA from these same two midbrain areas. SPA thresholds were measured with the tail-flick method and compared before and for up to two weeks after NRM lesions. A high positive correlation was found between percent NRM destruction and percent increase in SPA threshold for rats with ventral but not dorsal electrode placements. Damage to brain areas other than NRM seemed not to contribute to these effects. We conclude that n. raphe magnus is a critical relay in the pain-suppressive path from that area of the rat midbrain mediating an opioid form of stimulation-produced analgesia. INTRODUCTION We have recently reported the existence of qualitatively different forms of stimulation-produced analgesia (SPA) arising from adjacent regions of the rat midbrain a. One region comprised the midline caudal periaqueductal gray matter (PAG); the other, more ventrally, comprised the dorsal raphe nucleus within caudal P A G and the subjacent tegmentum. The most notable difference in SPA from these two areas was that the opiate antagonist naloxone reliably blocked analgesia only from the more ventral stimulation sites. On the other hand, stimulation of both dorsal and ventral loci inhibited the tail-flick reflex, known to be spinally mediated s, indicating that both areas have access to spinopetal paths. It is well established that these portions of the midbrain do not give rise to direct spinal projections in substantial numbers4,16. However, a good deal of evidence suggests that the bulbar n. raphe magnus ( N R M ) is an important relay between the midbrain
and the ultimate sites of pain suppression in the spinal cord dorsal horn 7. The N R M has been linked to both opioidg,10,1~ and non-opioid12,14 mechanisms of analgesia. In this study, therefore, we investigated whether discrete N R M lesions differentially affect analgesia from the naloxone-sensitive ventral versus the naloxone-insensitive dorsal SPA regions of the rat midbrain. METHODS Forty-six male, Sprague-Dawley rats (350450 g) were implanted with single, twisted bipolar stimulating electrodes (175 /~m diameter) under pentobarbital anesthesia. Electrodes were construtted of Teflon-coated stainless steel wires cut to equal length and bared of insulation at the crosssection of their tips. Using the atlas of Pellegrino and Cushman 11, electrodes were directed in the midline and at the same rostro-caudal level to either P A G sites above the dorsal raphe nucleus (dorsal
* Present address: Dcpartamento de Fisiologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico 20 D.F., Mexico. ** Present address: Department of Psychology, University of Scranton, Scranton, PA 15810, U.S.A. *** To whom correspondence should be addressed. 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
54 group) or to sites within or just below the dorsal raphe nucleus (ventral group). Animals were maintained on a 12 h light/dark cycle and had food and water available at all times. All testing occurred in the dark phase of the cycle. One week after implantation surgery the animals were handled and adapted to the test environment during ~ne or two one-hour sessions. For this and subsequent testing, animals were placed in Plexiglas tubes from which their tails protruded. SPA was assessed by means of the tail-flick test 6. The heat source was adjusted to elicit baseline response latencies in the 3.5-4.5 s range, and a cut-off time of 8.0 s was used for automatic termination of the heat to prevent tissue damage in analgesic animals. SPA threshold was defined as the current level necessary to inhibit the tail-flick response at this cut-off value. Two min separated all tail-flick trials. Brain stimulation preceded by 10 s and continued throughout a tail-flick trial. The stimulation consisted of constant current, rectangular, biphasic, pulse-pairs (20/s) delivered by a BERL 220B stimulator. Each pulse was 50/is in length with a 100 #s interpulse interval. The maximum current delivered was 18 mA peak-to-peak. Animals exhibiting substantial motor responses or clear signs of distress (escape attempts or vocalization) were excluded from the study. These reactions were rare and, as previously reported 3, tended to occur more frequently with stimulation of the dorsal region. At the start of an experimental session, baseline responsiveness on the tail-flick test was assessed by determining the mean response latency for the last 3 of 5 trials. Following this, thresholds for SPA were determined using a modified ascending method of limits. Brain stimulation was begun at 1 mA peakto-peak and increased in 1 mA steps for each subsequent tail-flick trial until a current level was reached that completely inhibited the tail-flick response. Stimulation intensity was then reduced by 0.8 mA, and testing continued using 0.2 m A increments until the tail-flick response was again inhibited. At this time, stimulation was stopped and tail-flick testing continued until the animal's response latencies returned to within the range of those occurring in the baseline series. Brain stimulation was then delivered at the last tested intensity. If analgesia was produced, this current level was defined as the SPA threshold.
If a tail-flick response occurred, however, threshold determinations were resumed until this criterion was attained. Animals were tested in the above manner twice at one week intervals. They were then anesthetized as before and received electrolytic lesions in the region of the N R M . These lesions were produced by passing 0.8 mA anodal constant DC current (5 s duration) at one or two midline points 0.8 m m below and 1.5-2.5 m m caudal to ear bar zero. For this surgery, the stereotaxic incisor bar was set level with the ear bars. Following a one-week recovery period, the animals were tested for SPA thresholds, as described above, once a week for 2 weeks. Assessments of lesion effects were made by comparing the mean SPA thresholds across the determinations made preceding and following these lesions. Determination of electrode locations and measurements of lesion damage were made using Nissl stained materials (60 /zm frozen sections) by an investigator who was unaware of the animals' experimental histories. Stimulating electrodes were designated as being either within the dorsal or ventral stimulation region. Within each region, the variation among electrode placements was slight and occurred within an area that we previously found to be homogenous for the production of SPA 3. The lesions were traced; the tracings were then cut out and weighed and the resulting data used in computing both percent N R M destruction and total lesion volume. RESULTS AND DISCUSSION The amount of N R M destruction was highly positively correlated with post-lesion elevations in SPA thresholds for animals with ventral stimulation electrode placements (n = 30, r = 0.81, P <: 0.01) (see Fig. 1). Although they sustained comparable amounts of N R M damage (varying between 2097 ~ destruction), animals with dorsal stimulation electrode placements (n = 17) exhibited no significant correlation between the amount of N R M damage and SPA threshold changes (r = 0.21, P 0.05). There was no significant correlation between lesion size and SPA threshold changes for either stimulation group after the effect of N R M damage was partialled out (r -- 0.15, dorsal group; r
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% Destruclion of n. Raphe Magnus Fig. 1. Scatter plot showing the relationship between percent destruction of n. raphe magnus and percent change in SPA thresholds for xats with ventral stimulation electrode placements. These data (n = 30) yield a significant positive correlation (r = 0.81, P < 0.01).
---0.20, ventral group), suggesting that destruction of other bulbar regions did not contribute significantly to the threshold elevations we observed. Moreover, partialling out the effect of lesion size from the relationship between N R M damage and threshold change neither eliminated the positive correlation between these measures for the ventral group (r = 0.72, P < 0.01) nor caused a significant correlation to occur for the dorsal group (r = 0.01, P > 0.05). The lesion depicted in Fig. 2 (91 ~ destruction of NRM) is from a typical animal with a ventral stimulating electrode that exhibited a marked elevation of SPA threshold post-operatively (147 ~). It is clear from this figure that effective lesions extended beyond the lateral border of the traditionally defined N R M L This fact leaves open the possibility that damage to proximal lateral structures, notably the n. paragigantocellularisl,~,14,17, may have contributed to the elevations in SPA thresholds that we found. Dividing the ventral stimulation group into those animals having damage primarily in the rostral half o f N R M (n -- 5) and those having damage primarily in the caudal half (n = 6) did not reveal any significant difference in the elevations in SPA thresholds
(P :> 0.05, Student's t-test). Because the n. paragigantocellularis is primarily associated with the caudal part of N R M 15, this analysis suggests that this adjoining medullary nucleus is not critically involved in the lesion effects. By the same reasoning, it would appear that extensions of lesions into raphe nuclei located just rostral or caudal to the N R M are also not critical for the lesion effects. Taken together with the partial correlation findings discussed above, we believe that our histological assessments point to the amount of N R M destruction being the principal factor in elevating SPA thresholds from our ventral stimulation region. In relation to SPA from dorsal stimulation placements, assessment of the effects of these midline lesions did not provide any indication of possible bulbar relays by which this region of the PAG exerts its spinal analgesic action. Only one animal in this group exhibited a substantial post-lesion shift in
Fig. 2. Representation of a large n. raphe magnus lesion (91 ~ destruction) from a rat showing a marked post-lesion elevation (147~) in SPA threshold. The lesion is shown by the stippled area in 6 frontal planes on diagrams modified from the stereotaxic atlas of Pellegrino and Cushman11.
56 SPA. This animal's lesion was not exceptional with regard to either percent N R M destruction or total lesion size. Overall, the post-lesion increases in SPA thresholds for animals with dorsally located electrodes were not significantly different from those of animals with ventral electrodes that had received less than 7 0 ~ destruction of the NRM. Proudfit 13 reported that the effects of NRM lesions on morphine analgesia change as a function of time post-lesion. We found no evidence for systematic alterations in lesion effects on SPA over the 2 week post-lesion interval examined. Additionally, the same author found that N R M lesions cause alterations in baseline tail-flick latencies 1~. We found no significant correlation between either lesion size or percentage of N R M destroyed and changes in baseline tail-flick latencies. Even when only considering the most extensively lesioned animals ( > 7 0 ~ destruction, n = 17), there were no significant differences in pre- versus post-lesion tailflick baselines (P > 0.05, Student's t-test). The reasons for these discrepancies are not clear. Our results are in general agreement with those of Behbehani and Fields 2 who reported that lesions in the region of the N R M disrupt the spinal analgesic action of glutamate injections into the PAG of the rat. There are some notable differences, however. We find that N R M lesions disrupt SPA from stimulation sites in the dorsal raphe n. and subjacent tegmentum, but not from electrode sites more dorsally in the midline caudal PAG. Behbehani and Fields 2 found that N R M lesions could disrupt analgesia derived from the PAG. Additionally, these authors concluded that lesions need encompass the N R M as well as areas lying just lateral to it in order to disrupt analgesia effectively. Our results suggest that if lesions destroy the rostro-caudal extent of NRM, this is sufficient to elevate SPA thresholds from the ventral stimulation region. There are two differences between our study and that of Behbehani and Fields 2 that offer possible resolutions to these discrepancies. The most obvious procedural difference is the means by which the midbrain was stimulated (i.e., chemically versus electrically). These two
approaches could involve different neural populations in the production of analgesia from the same stimulation loci. Electrical stimulation would be expected to affect all neural elements in the vicinity of the electrode tip, including, for example, axon terminals and fibers of passage. By contrast, glutamate is thought to excite neuronal cell bodies relatively selectively. Also, Behbehani and Fields 2 primarily examined injection sites in the ventrolateral aspect of the PAG, a locus we have not investigated. In our recent work 3 assessing the antagonistic action of naloxone on SPA from the same dorsal and ventral regions of the midbrain studied in the present report, we cautioned against generalizing our findings from the midline to the lateral aspects of PAG. This concern seems no less appropriate here. As mentioned earlier, the N R M has been associated with both opioid 9,1°,a7 and non-opioidl2,14 analgesic mechanisms. Our evidence suggests that the N R M is involved only in that type of SPA Ifrom ventral stimulation sites) found in our previous study 3 to be sensitive to naloxone blockade and hence presumably opioid mediated. On the other hand, it should be noted that the mean elevation in SPA threshold caused by large N R M lesions ( ~ 70 ~ destruction) was substantially greater than that caused by large doses ofnaloxone (10 mg/kg) in our previous study 3, 129 vs 3 7 ~ . The relatively weak naloxone effect appears to indicate that even within the ventral SPA area we have studied, both opioid and non-opioid SPA substrates exist. Such findings reinforce our earlier suggestion z that multiple, neurochemically and anatomically distinct endogenous analgesia systems reside within the medial midbrain of tile rat. ACKNOWLEDGEMENTS We are grateful to Mr. Thomas McGreal for his professional management of our vivarium service. Our research was supported by N I H grant NS07628. G.J.P. was supported by a grant from CONACYTMexico.
57 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 Behbehani, M. M. and Fields, H. L., Evidence that an excitatory connection between the periaqueductal gray and nucleus raphe magnus mediates stimulation produced analgesia, Brain Research, 170 (1979) 85-93. 3 Cannon, J. T., Prieto, G. J., Lee, A. and Liebeskind, J. C., Evidence for opioid and non-opioid forms of stimulation-produced analgesia in the rat, Brain Research, 243 (1982) 315-321. 4 Castiglioni, A. J., Gallaway, M. C. and Coulter, J. D., Spinal projections from the midbrain in monkey. J. comp. Neurol., 178 (1978) 329-346. 5 Dahlstr6m, 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 brain stem neurons, Acta. physiol, scand., 62, Suppl. 232 (1964) 1-55. 6 D'Amour, F. E. and Smith, D. L., A method for determining loss of pain sensation, J. PharmacoL exp. Ther., 72 (1941) 74-79. 7 Fields, H. L. and Basbaum, A. I., Anatomy and physiology of a descending pain control system. In J. J. Bonita, J. C. Liebeskind and D. G. Albe-Fessard (Eds.), Advances in Pain Research and Therapy, VoL 3, Raven Press, New York, 1979, pp. 427-440. 8 Irwin, S., Houde, R. W., Bennett, D. R., Hendershot, L. C. and Seevers, M. H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. Pharmacol. exp. Ther., 101 (1951) 132-143.
90leson, T. D., Twombly, D. A. and Liebeskind, J. C., Effects of pain-attenuating brain stimulation and morphine on electrical activity in the raphe nuclei of the awake rat, Pain, 4 (1978) 211-230. 10 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 (1975) 139-145. 11 Pellegrino, L. J. and Cushman, A. J., A Stereotaxic Atlas of the Rat Brain, Appleton-Century-Crofts, New York, 1967. 12 Proudfit, H. K., Reversible inactivation of raphe magnus neurons: effects on nociceptive threshold and morphineinduced analgesia, Brain Research, 201 (1980) 459-464. 13 Proudfit, H. K., Time-course of alterations in morphineinduced analgesia and nociceptive threshold following medullary raphe lesions, Neuroscience, 6 (1981) 945-951. 14 Satoh, M., Akaike, A., Nakazawa, T. and Takagi, H., Evidence for involvement of separate mechanisms in the production of analgesia by electrical stimulation of the nucleus reticularis paragigantocellularis and nucleus raphe magnus in the rat, Brain Research, 194 (1980) 525-529. 15 Taber, E., Brodal, A. and Walberg, F., The raphe nuclei of the brain stem in the cat. I. Normal topography and cytoarchitecture and general discussion, J. comp. Neurol., 114 (1960) 161-182. 16 Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., Identification and somatotopic organization of nuclei projecting via the dorsolateral funiculus in rats: a retrograde tracing study using HRP slow-release gels, Brain Research, 223 (1981) 237-255. 17 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.
53
N. Raphe Magnus Lesions Disrupt Stimulation-Produced Analgesia from Ventral but not Dorsal Midbrain Areas in the Rat G. J. PRIETO*, J. T. CANNON** and J. C. LIEBESKIND*** Department of Psychology, UCLA, Los Angeles, CA 90024, (U.S.A.)
(Accepted July 20th, 1982) Key words: stimulation-produced analgesia - - pain - - analgesia - - n. raphe magnus - periaqueductal gray matter - - opioid peptides
We previously found that the opiate antagonist, naloxone, partially blocks stimulation-produced analgesia (SPA) elicited from ventral but not dorsal regions of the medial midbrain in rats. The present study compares the effects of n. raphe magnus (NRM) lesions on SPA from these same two midbrain areas. SPA thresholds were measured with the tail-flick method and compared before and for up to two weeks after NRM lesions. A high positive correlation was found between percent NRM destruction and percent increase in SPA threshold for rats with ventral but not dorsal electrode placements. Damage to brain areas other than NRM seemed not to contribute to these effects. We conclude that n. raphe magnus is a critical relay in the pain-suppressive path from that area of the rat midbrain mediating an opioid form of stimulation-produced analgesia. INTRODUCTION We have recently reported the existence of qualitatively different forms of stimulation-produced analgesia (SPA) arising from adjacent regions of the rat midbrain a. One region comprised the midline caudal periaqueductal gray matter (PAG); the other, more ventrally, comprised the dorsal raphe nucleus within caudal P A G and the subjacent tegmentum. The most notable difference in SPA from these two areas was that the opiate antagonist naloxone reliably blocked analgesia only from the more ventral stimulation sites. On the other hand, stimulation of both dorsal and ventral loci inhibited the tail-flick reflex, known to be spinally mediated s, indicating that both areas have access to spinopetal paths. It is well established that these portions of the midbrain do not give rise to direct spinal projections in substantial numbers4,16. However, a good deal of evidence suggests that the bulbar n. raphe magnus ( N R M ) is an important relay between the midbrain
and the ultimate sites of pain suppression in the spinal cord dorsal horn 7. The N R M has been linked to both opioidg,10,1~ and non-opioid12,14 mechanisms of analgesia. In this study, therefore, we investigated whether discrete N R M lesions differentially affect analgesia from the naloxone-sensitive ventral versus the naloxone-insensitive dorsal SPA regions of the rat midbrain. METHODS Forty-six male, Sprague-Dawley rats (350450 g) were implanted with single, twisted bipolar stimulating electrodes (175 /~m diameter) under pentobarbital anesthesia. Electrodes were construtted of Teflon-coated stainless steel wires cut to equal length and bared of insulation at the crosssection of their tips. Using the atlas of Pellegrino and Cushman 11, electrodes were directed in the midline and at the same rostro-caudal level to either P A G sites above the dorsal raphe nucleus (dorsal
* Present address: Dcpartamento de Fisiologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico 20 D.F., Mexico. ** Present address: Department of Psychology, University of Scranton, Scranton, PA 15810, U.S.A. *** To whom correspondence should be addressed. 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
54 group) or to sites within or just below the dorsal raphe nucleus (ventral group). Animals were maintained on a 12 h light/dark cycle and had food and water available at all times. All testing occurred in the dark phase of the cycle. One week after implantation surgery the animals were handled and adapted to the test environment during ~ne or two one-hour sessions. For this and subsequent testing, animals were placed in Plexiglas tubes from which their tails protruded. SPA was assessed by means of the tail-flick test 6. The heat source was adjusted to elicit baseline response latencies in the 3.5-4.5 s range, and a cut-off time of 8.0 s was used for automatic termination of the heat to prevent tissue damage in analgesic animals. SPA threshold was defined as the current level necessary to inhibit the tail-flick response at this cut-off value. Two min separated all tail-flick trials. Brain stimulation preceded by 10 s and continued throughout a tail-flick trial. The stimulation consisted of constant current, rectangular, biphasic, pulse-pairs (20/s) delivered by a BERL 220B stimulator. Each pulse was 50/is in length with a 100 #s interpulse interval. The maximum current delivered was 18 mA peak-to-peak. Animals exhibiting substantial motor responses or clear signs of distress (escape attempts or vocalization) were excluded from the study. These reactions were rare and, as previously reported 3, tended to occur more frequently with stimulation of the dorsal region. At the start of an experimental session, baseline responsiveness on the tail-flick test was assessed by determining the mean response latency for the last 3 of 5 trials. Following this, thresholds for SPA were determined using a modified ascending method of limits. Brain stimulation was begun at 1 mA peakto-peak and increased in 1 mA steps for each subsequent tail-flick trial until a current level was reached that completely inhibited the tail-flick response. Stimulation intensity was then reduced by 0.8 mA, and testing continued using 0.2 m A increments until the tail-flick response was again inhibited. At this time, stimulation was stopped and tail-flick testing continued until the animal's response latencies returned to within the range of those occurring in the baseline series. Brain stimulation was then delivered at the last tested intensity. If analgesia was produced, this current level was defined as the SPA threshold.
If a tail-flick response occurred, however, threshold determinations were resumed until this criterion was attained. Animals were tested in the above manner twice at one week intervals. They were then anesthetized as before and received electrolytic lesions in the region of the N R M . These lesions were produced by passing 0.8 mA anodal constant DC current (5 s duration) at one or two midline points 0.8 m m below and 1.5-2.5 m m caudal to ear bar zero. For this surgery, the stereotaxic incisor bar was set level with the ear bars. Following a one-week recovery period, the animals were tested for SPA thresholds, as described above, once a week for 2 weeks. Assessments of lesion effects were made by comparing the mean SPA thresholds across the determinations made preceding and following these lesions. Determination of electrode locations and measurements of lesion damage were made using Nissl stained materials (60 /zm frozen sections) by an investigator who was unaware of the animals' experimental histories. Stimulating electrodes were designated as being either within the dorsal or ventral stimulation region. Within each region, the variation among electrode placements was slight and occurred within an area that we previously found to be homogenous for the production of SPA 3. The lesions were traced; the tracings were then cut out and weighed and the resulting data used in computing both percent N R M destruction and total lesion volume. RESULTS AND DISCUSSION The amount of N R M destruction was highly positively correlated with post-lesion elevations in SPA thresholds for animals with ventral stimulation electrode placements (n = 30, r = 0.81, P <: 0.01) (see Fig. 1). Although they sustained comparable amounts of N R M damage (varying between 2097 ~ destruction), animals with dorsal stimulation electrode placements (n = 17) exhibited no significant correlation between the amount of N R M damage and SPA threshold changes (r = 0.21, P 0.05). There was no significant correlation between lesion size and SPA threshold changes for either stimulation group after the effect of N R M damage was partialled out (r -- 0.15, dorsal group; r
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% Destruclion of n. Raphe Magnus Fig. 1. Scatter plot showing the relationship between percent destruction of n. raphe magnus and percent change in SPA thresholds for xats with ventral stimulation electrode placements. These data (n = 30) yield a significant positive correlation (r = 0.81, P < 0.01).
---0.20, ventral group), suggesting that destruction of other bulbar regions did not contribute significantly to the threshold elevations we observed. Moreover, partialling out the effect of lesion size from the relationship between N R M damage and threshold change neither eliminated the positive correlation between these measures for the ventral group (r = 0.72, P < 0.01) nor caused a significant correlation to occur for the dorsal group (r = 0.01, P > 0.05). The lesion depicted in Fig. 2 (91 ~ destruction of NRM) is from a typical animal with a ventral stimulating electrode that exhibited a marked elevation of SPA threshold post-operatively (147 ~). It is clear from this figure that effective lesions extended beyond the lateral border of the traditionally defined N R M L This fact leaves open the possibility that damage to proximal lateral structures, notably the n. paragigantocellularisl,~,14,17, may have contributed to the elevations in SPA thresholds that we found. Dividing the ventral stimulation group into those animals having damage primarily in the rostral half o f N R M (n -- 5) and those having damage primarily in the caudal half (n = 6) did not reveal any significant difference in the elevations in SPA thresholds
(P :> 0.05, Student's t-test). Because the n. paragigantocellularis is primarily associated with the caudal part of N R M 15, this analysis suggests that this adjoining medullary nucleus is not critically involved in the lesion effects. By the same reasoning, it would appear that extensions of lesions into raphe nuclei located just rostral or caudal to the N R M are also not critical for the lesion effects. Taken together with the partial correlation findings discussed above, we believe that our histological assessments point to the amount of N R M destruction being the principal factor in elevating SPA thresholds from our ventral stimulation region. In relation to SPA from dorsal stimulation placements, assessment of the effects of these midline lesions did not provide any indication of possible bulbar relays by which this region of the PAG exerts its spinal analgesic action. Only one animal in this group exhibited a substantial post-lesion shift in
Fig. 2. Representation of a large n. raphe magnus lesion (91 ~ destruction) from a rat showing a marked post-lesion elevation (147~) in SPA threshold. The lesion is shown by the stippled area in 6 frontal planes on diagrams modified from the stereotaxic atlas of Pellegrino and Cushman11.
56 SPA. This animal's lesion was not exceptional with regard to either percent N R M destruction or total lesion size. Overall, the post-lesion increases in SPA thresholds for animals with dorsally located electrodes were not significantly different from those of animals with ventral electrodes that had received less than 7 0 ~ destruction of the NRM. Proudfit 13 reported that the effects of NRM lesions on morphine analgesia change as a function of time post-lesion. We found no evidence for systematic alterations in lesion effects on SPA over the 2 week post-lesion interval examined. Additionally, the same author found that N R M lesions cause alterations in baseline tail-flick latencies 1~. We found no significant correlation between either lesion size or percentage of N R M destroyed and changes in baseline tail-flick latencies. Even when only considering the most extensively lesioned animals ( > 7 0 ~ destruction, n = 17), there were no significant differences in pre- versus post-lesion tailflick baselines (P > 0.05, Student's t-test). The reasons for these discrepancies are not clear. Our results are in general agreement with those of Behbehani and Fields 2 who reported that lesions in the region of the N R M disrupt the spinal analgesic action of glutamate injections into the PAG of the rat. There are some notable differences, however. We find that N R M lesions disrupt SPA from stimulation sites in the dorsal raphe n. and subjacent tegmentum, but not from electrode sites more dorsally in the midline caudal PAG. Behbehani and Fields 2 found that N R M lesions could disrupt analgesia derived from the PAG. Additionally, these authors concluded that lesions need encompass the N R M as well as areas lying just lateral to it in order to disrupt analgesia effectively. Our results suggest that if lesions destroy the rostro-caudal extent of NRM, this is sufficient to elevate SPA thresholds from the ventral stimulation region. There are two differences between our study and that of Behbehani and Fields 2 that offer possible resolutions to these discrepancies. The most obvious procedural difference is the means by which the midbrain was stimulated (i.e., chemically versus electrically). These two
approaches could involve different neural populations in the production of analgesia from the same stimulation loci. Electrical stimulation would be expected to affect all neural elements in the vicinity of the electrode tip, including, for example, axon terminals and fibers of passage. By contrast, glutamate is thought to excite neuronal cell bodies relatively selectively. Also, Behbehani and Fields 2 primarily examined injection sites in the ventrolateral aspect of the PAG, a locus we have not investigated. In our recent work 3 assessing the antagonistic action of naloxone on SPA from the same dorsal and ventral regions of the midbrain studied in the present report, we cautioned against generalizing our findings from the midline to the lateral aspects of PAG. This concern seems no less appropriate here. As mentioned earlier, the N R M has been associated with both opioid 9,1°,a7 and non-opioidl2,14 analgesic mechanisms. Our evidence suggests that the N R M is involved only in that type of SPA Ifrom ventral stimulation sites) found in our previous study 3 to be sensitive to naloxone blockade and hence presumably opioid mediated. On the other hand, it should be noted that the mean elevation in SPA threshold caused by large N R M lesions ( ~ 70 ~ destruction) was substantially greater than that caused by large doses ofnaloxone (10 mg/kg) in our previous study 3, 129 vs 3 7 ~ . The relatively weak naloxone effect appears to indicate that even within the ventral SPA area we have studied, both opioid and non-opioid SPA substrates exist. Such findings reinforce our earlier suggestion z that multiple, neurochemically and anatomically distinct endogenous analgesia systems reside within the medial midbrain of tile rat. ACKNOWLEDGEMENTS We are grateful to Mr. Thomas McGreal for his professional management of our vivarium service. Our research was supported by N I H grant NS07628. G.J.P. was supported by a grant from CONACYTMexico.
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