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Microinjection of morphine into nucleus reticularis paragigantocellularis of the rat: Suppression of noxious-evoked activity of nucleus raphe magnus neurons
388
Brain Research, 359 (1985) 388-391 Elsevier
BRE 21245
M~injection of m o ~ into n u ~ u s r~ioularis paragigantoceilularis of the rat: supprmmion of noxle,u~-ev~d(ed s~ivity of nucleus r a p ~ m ~ u s neurons MARY M. HEINRICHER* and J. PETER ROSENFELD Northwestern University, Departments of Neurobiology/Physiology and Psychology, Cresap Laboratory, Evanston, IL 60201 (U.S.A.) (Accepted August 6th, 1985) Key words: pain - - analgesia - - nucleus raphe magnus --nucleus reticularis paragigantocellularis- - morphine m microinjection
Microinjection of 1#g of morphine into nucleus reticularis paragigantocellularis (Pgc) of anesthetized rats depressed both noxiousevoked and spontaneous activity of nociresponsive neurons in the nucleus raphe magnus (NRM). This effect was naloxone-reversible, and was not observed after control injections dorsal to Pgc. The percentchange in spontaneous firing was significantlygreater than the percent change in pinch-evoked firing. This reduction in NRM neuronal discharge may contribute to the antinoeiceptive effects produced by microinjection of morphine into Pgc. Microinjection of morphine in the nucleus reticularis paragigantocellularis (Pgc) of the ventral medulla has antinociceptive effects 1,4,16. Although the mechanisms by which such microinjections result in analgesia are not yet understood, there is evidence for the involvement of the nucleus raphe magnus (NRM), since Pgc has reciprocal connections with NRM 3,8,9,11 and since electrolytic lesions of NRM block the analgesia produced by microinjection of large (5 ~g) doses of morphine into Pgc 4. Thus, it would be of interest to understand the functional relationships between Pgc and NRM. We have recently shown that microinjection of morphine into Pgc results in suppression of the spontaneous firing of the majority of spontaneously active, nociresponsive NRM neurons 15. In the experiments reported here, we have extended our observations to include the noxiousevoked activity of NRM cells. Twenty-three male Sprague-D awley-derived rats, 350-450 g, were used. They were anesthetized with urethane (1.2 g/kg) and placed in a stereotaxic instrument. Body temperature was maintained at 37 °C. Microinjection parameters were designed to dupli-
cate those used in behavioral experiments in this laboratory. A 25-gauge stainless steel guide cannula was implanted, aimed toward Pgc 13 (15 experiments) or toward the nucleus reticularis gigantoceUularis (NGC) (8 experiments). This cannula was lowered to a point 2 mm above the intended injection site. An injector cannula made of 31-gauge stainless steel tubing was inserted through the guide so that the injector tip projected 2 mm beyond the end of the guide. The injector was attached to a length of polyethylene tubing (PE 20) and microinjections were made using a 50-/A syringe and an infusion pump. Morphine sulfate (Lilly) was dissolved in isotonic saline and 1/~g was administered in a volume of 0.25 #l over 3 - 4 min. Single cell activity was recorded extracellularly from NRM using glass micropipettes filled with 0.5 M sodium acetate and 2% Pontamine Sky Blue. Unit activity was amplified and led to a window discriminator and ratemeter, an audio monitor and an oscilloscope. The noxious stimulus used in these experiments consisted of a strong pinch delivered using a pair of pliers with toothed jaws. A stop was inserted between the jaws so that the distance between the tips
* Present address: Department of Physiology, School of Medicine, University of California, San Francisco, CA 94143, U.S.A. Correspondence: J.P. Rosenfeld, Northwestern University, Departments of Neurobiology/Physiologyand Psychology, Cresap Laboratory, Evanston, IL 60201, U.S.A.
389 when the device was fully closed remained constant. The area of skin to be pinched was marked so that the same amount of skin was between the jaws on each pinch. An audible signal provided by computer was used to control the duration of the pinch. After isolating a cell which was excited by noxious pinch, a premorphine measure of neuronal activity was taken. Evoked firing rate was defined as the average firing rate (in spikes/s) during 3-5 repetitions of a 5-s pinch. Spontaneous firing rate was defined as the average firing rate (in spikes/s) over a 5-s period preceding each noxious pinch. Morphine (1/~g) was then microinjected into Pgc, and the spontaneous and noxious-evoked discharge of the cell was observed for the following 40-60 min. The locations of the recording electrodes and injector cannulae were histologically verified. The recording electrode was found to be within NRM for all experiments. All cells described in this report were both spontaneously active and excited by noxious pinch. Spontaneous activity ranged from 3.01 to 44.37 Hz (mean: 18.94 Hz). Receptive fields for pinch were large, often including both the face and lower body. Approxi-
mately one-half of the cells were also excited by nonnoxious tactile stimulation, although this response was smaller than that elicited by noxious stimulation. Morphine was microinjected into Pgc in 15 experiments. This resulted in a decrease in the spontaneous activity of all 15 NRM neurons. These cells showed a mean 53.6% decrease in firing rate measured at 10-15 min after the microinjection (range: 23.6-99%). Morphine microinjection was followed by a mean 23.7% decrease in pinch-evoked activity (range: 0-76%). In 3 cases, pinch-evoked activity was unchanged following microinjection of morphine into Pgc. In the 13 experiments in which it was administered, naloxone (1 mg/kg) partially (10 cells) or completely (3 cells) reversed the morphine-induced suppression. A typical neuron is shown in Fig. 1. Microinjection sites are shown in Fig. 2. In order to determine if the effect of morphine on noxious-evoked activity was different from that on spontaneous activity, a difference score was obtained for each cell by subtracting the percent change in pinch-evoked firing from the percent change in spontaneous firing. This difference score was then used in
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NAL Fig. 1. Microinjection of morphine into Pgc resulted in a 70% decrease in spontaneous activity. The change in pinch-evoked activity was 17%. Naloxone attenuated the suppression of spontaneous activity. Pinch delivered at time indicated by underscore. Calibration: 100 s, 10 spikes/s.
390
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Fig. 2. Locations of cannula tips in experiments in which morphine microinjection resulted in decreased activity of NRM neuron (filled circles) or in no effect on NRM firing (open triangles), amb, nucleus ambiguus; io, inferior olive; ngc, n. reticularis gigantocellularis; pgc, n. reticularis paragigantocellularis; VII, nucleus of VII nerve. a two-tailed correlated-means t-test, which showed that the suppression of noxious-evoked activity was significantly less than that of spontaneous discharge (t = 5.5 (14), P < 0.01). In 6 cases, morphine was microinjected into N G C and in 2 cases into the VII nerve nucleus. These microinjections had no significant effect on spontaneous or noxious-evoked firing of N R M neurons. The results of these experiments replicate our earlier finding of suppression of the spontaneous discharge of N R M neurons following microinjection of an antinociceptive dose of morphine into Pgc 15. In addition, they demonstrate that the spontaneous firing of these neurons is also suppressed, and that noxious-evoked activity is depressed to a lesser degree
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 Anderson, E.G., Lobatz, M. and Proudfit, H.K., The effects of pain and opiates on unit activity in the nucleus raphe magnus. In R.W. Ryall and J.S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, Elsevier, Amsterdam, 1978, pp. 299-301.
than is ongoing background activity. The suppression of N R M activity was attenuated by a low systemic dose of naloxone, which suggests that this was a specific opiate effect and was not due to the microinjection procedures per se. The changes in N R M firing were also unlikely to be due to spread of the opiate to N R M itself, inasmuch as control microinjections dorsal to Pgc did not result in significant alterations in firing, and because the microinjection effects were different from those observed after direct iontophoretic application of opiates to N R M neurons 2,14. The lack of effect of N G C morphine microinjections on N R M activity is consistent with the suggestion that Pgc is more sensitive to microinjected opiates than is NGCJ. The neuronal circuitry responsible for the observed effects on N R M activity was not investigated in our experiments, but anatomical considerations suggest that Pgc may influence N R M activity directly and/or indirectly. Direct serotonin-, enkephalin-, neurotensin- and substance P-containing projections from Pgc to N R M have been identifiedS, 9 and one or more of these putative neurotransmitters may mediate the effects of Pgc morphine microinjections. Pgc morphine microinjections may also alter activity in other structures which have connections with NRM, such as the midbrain periaqueductal gray (PAG) 8,9.17. Anatomical investigations have documented projections from Pgc to the P A G 10. which has been shown in electrophysiological experiments to influence the activity of N R M neurons6,7.12. It is also possible that microinjections of morphine into Pgc could activate reticulospinal pathways originating in Pgc 5, which could result in altered peripheral input to NRM. Supported by N I H Grants G M 23696 and D E 05204.
3 Andrezik, J.A., Chan-Palay, V. and Palay, S.L., The nucleus paraglgantocellularis lateralis in the rat. Demonstration of afferents by the retrograde transport of horseradish peroxidase. Anat. Histol. Embryol., 161 (1981) 373-390. 4 Azami, J., Llewelyn, M.B. and Roberts, M.H.T.. The contribution of nucleus reticularis paragigantocellularis and nucleus raphe magnus to the analgesia produced by systemically administered morphine, investigated with the microinjection technique, Pain, 12 (1982) 229-246. 5 Basbaum, A.I. and Fields. H.L. The origin of descending
391
6
7
8
9
10
11
pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. Comp. Neurol., 187 (1979) 513-532. 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. Behbehani, M.M. and Pomeroy, S.L., Effect of morphine injected in periaqueductal gray on the activity of single units in nucleus raphe magnus of the rat, Brain Research, 149 (1978) 266-269. Beitz, A.J., The nuclei of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus, Neuroscience, 7 (1982) 2753-2768. Beitz, A.J., The sites of origin of brain stem neurotensin and serotonin projections to the rodent nucleus raphe magnus, J. Neurosci., 2 (1982) 829-842. Beitz, A.J., The organization of afferent projections to the midbrain periaqueductal gray of the rat, Neuroscience, 7 (1982) 133-159. Carlton, S.M., Leichnetz, G.R., Young, E.G. and Mayer, D.J., Supramedullary afferents of the nucleus raphe magnus in the rat: a study using the transcannula HRP gel and autoradiographic techniques, J. Comp. Neurol., 214 (1983) 43-58.
12 Fields, H.L. and Anderson, S.D., Evidence that raphe-spinal neurons mediate opiate and midbrain stimulation-produced analgesias, Pain, 5 (1978) 333-349. 13 Fifkov~i, E. and MarSala, J., Stereotaxic atlases for the cat, rabbit, and rat. In J. Buret, M. Petr~fi and J. Zacher (Eds.), Electrophysiological Methods in Biological Research, Academia, Prague, 1967, pp. 653-671. 14 Haigler, H.J., Morphine: effects on brainstem neurons. In R.W. Ryall and J.S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, Elsevier, Amsterdam, 1978. 15 Heinricher, M.M. and Rosenfeld, J.P., Microinjection of morphine into nucleus reticularis paragigantocellularis of the rat suppresses spontaneous activity of nucleus raphe magnus neurons, Brain Research, 272 (1983) 382-386. 16 Rosenfeld, J.P. and Stocco, S., Differential effects of systemic versus intracranial injection of opiates on central, orofacial, and lower body nociception: somatotypy in bulbar analgesia systems, Pain, 9 (1980) 307-318. 17 Shah, Y. and Dostrovsky, J.P., Electrophysiological evidence for a projection of the periaqueductal gray matter to nucleus raphe magnus in cat and rat, Brain Research, 193 (1980) 534-538.
Brain Research, 359 (1985) 388-391 Elsevier
BRE 21245
M~injection of m o ~ into n u ~ u s r~ioularis paragigantoceilularis of the rat: supprmmion of noxle,u~-ev~d(ed s~ivity of nucleus r a p ~ m ~ u s neurons MARY M. HEINRICHER* and J. PETER ROSENFELD Northwestern University, Departments of Neurobiology/Physiology and Psychology, Cresap Laboratory, Evanston, IL 60201 (U.S.A.) (Accepted August 6th, 1985) Key words: pain - - analgesia - - nucleus raphe magnus --nucleus reticularis paragigantocellularis- - morphine m microinjection
Microinjection of 1#g of morphine into nucleus reticularis paragigantocellularis (Pgc) of anesthetized rats depressed both noxiousevoked and spontaneous activity of nociresponsive neurons in the nucleus raphe magnus (NRM). This effect was naloxone-reversible, and was not observed after control injections dorsal to Pgc. The percentchange in spontaneous firing was significantlygreater than the percent change in pinch-evoked firing. This reduction in NRM neuronal discharge may contribute to the antinoeiceptive effects produced by microinjection of morphine into Pgc. Microinjection of morphine in the nucleus reticularis paragigantocellularis (Pgc) of the ventral medulla has antinociceptive effects 1,4,16. Although the mechanisms by which such microinjections result in analgesia are not yet understood, there is evidence for the involvement of the nucleus raphe magnus (NRM), since Pgc has reciprocal connections with NRM 3,8,9,11 and since electrolytic lesions of NRM block the analgesia produced by microinjection of large (5 ~g) doses of morphine into Pgc 4. Thus, it would be of interest to understand the functional relationships between Pgc and NRM. We have recently shown that microinjection of morphine into Pgc results in suppression of the spontaneous firing of the majority of spontaneously active, nociresponsive NRM neurons 15. In the experiments reported here, we have extended our observations to include the noxiousevoked activity of NRM cells. Twenty-three male Sprague-D awley-derived rats, 350-450 g, were used. They were anesthetized with urethane (1.2 g/kg) and placed in a stereotaxic instrument. Body temperature was maintained at 37 °C. Microinjection parameters were designed to dupli-
cate those used in behavioral experiments in this laboratory. A 25-gauge stainless steel guide cannula was implanted, aimed toward Pgc 13 (15 experiments) or toward the nucleus reticularis gigantoceUularis (NGC) (8 experiments). This cannula was lowered to a point 2 mm above the intended injection site. An injector cannula made of 31-gauge stainless steel tubing was inserted through the guide so that the injector tip projected 2 mm beyond the end of the guide. The injector was attached to a length of polyethylene tubing (PE 20) and microinjections were made using a 50-/A syringe and an infusion pump. Morphine sulfate (Lilly) was dissolved in isotonic saline and 1/~g was administered in a volume of 0.25 #l over 3 - 4 min. Single cell activity was recorded extracellularly from NRM using glass micropipettes filled with 0.5 M sodium acetate and 2% Pontamine Sky Blue. Unit activity was amplified and led to a window discriminator and ratemeter, an audio monitor and an oscilloscope. The noxious stimulus used in these experiments consisted of a strong pinch delivered using a pair of pliers with toothed jaws. A stop was inserted between the jaws so that the distance between the tips
* Present address: Department of Physiology, School of Medicine, University of California, San Francisco, CA 94143, U.S.A. Correspondence: J.P. Rosenfeld, Northwestern University, Departments of Neurobiology/Physiologyand Psychology, Cresap Laboratory, Evanston, IL 60201, U.S.A.
389 when the device was fully closed remained constant. The area of skin to be pinched was marked so that the same amount of skin was between the jaws on each pinch. An audible signal provided by computer was used to control the duration of the pinch. After isolating a cell which was excited by noxious pinch, a premorphine measure of neuronal activity was taken. Evoked firing rate was defined as the average firing rate (in spikes/s) during 3-5 repetitions of a 5-s pinch. Spontaneous firing rate was defined as the average firing rate (in spikes/s) over a 5-s period preceding each noxious pinch. Morphine (1/~g) was then microinjected into Pgc, and the spontaneous and noxious-evoked discharge of the cell was observed for the following 40-60 min. The locations of the recording electrodes and injector cannulae were histologically verified. The recording electrode was found to be within NRM for all experiments. All cells described in this report were both spontaneously active and excited by noxious pinch. Spontaneous activity ranged from 3.01 to 44.37 Hz (mean: 18.94 Hz). Receptive fields for pinch were large, often including both the face and lower body. Approxi-
mately one-half of the cells were also excited by nonnoxious tactile stimulation, although this response was smaller than that elicited by noxious stimulation. Morphine was microinjected into Pgc in 15 experiments. This resulted in a decrease in the spontaneous activity of all 15 NRM neurons. These cells showed a mean 53.6% decrease in firing rate measured at 10-15 min after the microinjection (range: 23.6-99%). Morphine microinjection was followed by a mean 23.7% decrease in pinch-evoked activity (range: 0-76%). In 3 cases, pinch-evoked activity was unchanged following microinjection of morphine into Pgc. In the 13 experiments in which it was administered, naloxone (1 mg/kg) partially (10 cells) or completely (3 cells) reversed the morphine-induced suppression. A typical neuron is shown in Fig. 1. Microinjection sites are shown in Fig. 2. In order to determine if the effect of morphine on noxious-evoked activity was different from that on spontaneous activity, a difference score was obtained for each cell by subtracting the percent change in pinch-evoked firing from the percent change in spontaneous firing. This difference score was then used in
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NAL Fig. 1. Microinjection of morphine into Pgc resulted in a 70% decrease in spontaneous activity. The change in pinch-evoked activity was 17%. Naloxone attenuated the suppression of spontaneous activity. Pinch delivered at time indicated by underscore. Calibration: 100 s, 10 spikes/s.
390
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•
ngc
2rnb//j /
Fig. 2. Locations of cannula tips in experiments in which morphine microinjection resulted in decreased activity of NRM neuron (filled circles) or in no effect on NRM firing (open triangles), amb, nucleus ambiguus; io, inferior olive; ngc, n. reticularis gigantocellularis; pgc, n. reticularis paragigantocellularis; VII, nucleus of VII nerve. a two-tailed correlated-means t-test, which showed that the suppression of noxious-evoked activity was significantly less than that of spontaneous discharge (t = 5.5 (14), P < 0.01). In 6 cases, morphine was microinjected into N G C and in 2 cases into the VII nerve nucleus. These microinjections had no significant effect on spontaneous or noxious-evoked firing of N R M neurons. The results of these experiments replicate our earlier finding of suppression of the spontaneous discharge of N R M neurons following microinjection of an antinociceptive dose of morphine into Pgc 15. In addition, they demonstrate that the spontaneous firing of these neurons is also suppressed, and that noxious-evoked activity is depressed to a lesser degree
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 Anderson, E.G., Lobatz, M. and Proudfit, H.K., The effects of pain and opiates on unit activity in the nucleus raphe magnus. In R.W. Ryall and J.S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, Elsevier, Amsterdam, 1978, pp. 299-301.
than is ongoing background activity. The suppression of N R M activity was attenuated by a low systemic dose of naloxone, which suggests that this was a specific opiate effect and was not due to the microinjection procedures per se. The changes in N R M firing were also unlikely to be due to spread of the opiate to N R M itself, inasmuch as control microinjections dorsal to Pgc did not result in significant alterations in firing, and because the microinjection effects were different from those observed after direct iontophoretic application of opiates to N R M neurons 2,14. The lack of effect of N G C morphine microinjections on N R M activity is consistent with the suggestion that Pgc is more sensitive to microinjected opiates than is NGCJ. The neuronal circuitry responsible for the observed effects on N R M activity was not investigated in our experiments, but anatomical considerations suggest that Pgc may influence N R M activity directly and/or indirectly. Direct serotonin-, enkephalin-, neurotensin- and substance P-containing projections from Pgc to N R M have been identifiedS, 9 and one or more of these putative neurotransmitters may mediate the effects of Pgc morphine microinjections. Pgc morphine microinjections may also alter activity in other structures which have connections with NRM, such as the midbrain periaqueductal gray (PAG) 8,9.17. Anatomical investigations have documented projections from Pgc to the P A G 10. which has been shown in electrophysiological experiments to influence the activity of N R M neurons6,7.12. It is also possible that microinjections of morphine into Pgc could activate reticulospinal pathways originating in Pgc 5, which could result in altered peripheral input to NRM. Supported by N I H Grants G M 23696 and D E 05204.
3 Andrezik, J.A., Chan-Palay, V. and Palay, S.L., The nucleus paraglgantocellularis lateralis in the rat. Demonstration of afferents by the retrograde transport of horseradish peroxidase. Anat. Histol. Embryol., 161 (1981) 373-390. 4 Azami, J., Llewelyn, M.B. and Roberts, M.H.T.. The contribution of nucleus reticularis paragigantocellularis and nucleus raphe magnus to the analgesia produced by systemically administered morphine, investigated with the microinjection technique, Pain, 12 (1982) 229-246. 5 Basbaum, A.I. and Fields. H.L. The origin of descending
391
6
7
8
9
10
11
pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. Comp. Neurol., 187 (1979) 513-532. 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. Behbehani, M.M. and Pomeroy, S.L., Effect of morphine injected in periaqueductal gray on the activity of single units in nucleus raphe magnus of the rat, Brain Research, 149 (1978) 266-269. Beitz, A.J., The nuclei of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus, Neuroscience, 7 (1982) 2753-2768. Beitz, A.J., The sites of origin of brain stem neurotensin and serotonin projections to the rodent nucleus raphe magnus, J. Neurosci., 2 (1982) 829-842. Beitz, A.J., The organization of afferent projections to the midbrain periaqueductal gray of the rat, Neuroscience, 7 (1982) 133-159. Carlton, S.M., Leichnetz, G.R., Young, E.G. and Mayer, D.J., Supramedullary afferents of the nucleus raphe magnus in the rat: a study using the transcannula HRP gel and autoradiographic techniques, J. Comp. Neurol., 214 (1983) 43-58.
12 Fields, H.L. and Anderson, S.D., Evidence that raphe-spinal neurons mediate opiate and midbrain stimulation-produced analgesias, Pain, 5 (1978) 333-349. 13 Fifkov~i, E. and MarSala, J., Stereotaxic atlases for the cat, rabbit, and rat. In J. Buret, M. Petr~fi and J. Zacher (Eds.), Electrophysiological Methods in Biological Research, Academia, Prague, 1967, pp. 653-671. 14 Haigler, H.J., Morphine: effects on brainstem neurons. In R.W. Ryall and J.S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, Elsevier, Amsterdam, 1978. 15 Heinricher, M.M. and Rosenfeld, J.P., Microinjection of morphine into nucleus reticularis paragigantocellularis of the rat suppresses spontaneous activity of nucleus raphe magnus neurons, Brain Research, 272 (1983) 382-386. 16 Rosenfeld, J.P. and Stocco, S., Differential effects of systemic versus intracranial injection of opiates on central, orofacial, and lower body nociception: somatotypy in bulbar analgesia systems, Pain, 9 (1980) 307-318. 17 Shah, Y. and Dostrovsky, J.P., Electrophysiological evidence for a projection of the periaqueductal gray matter to nucleus raphe magnus in cat and rat, Brain Research, 193 (1980) 534-538.