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The role of serotonergic neurons in nucleus raphe magnus in opioid antinociception
Commentary
The Role of Serotonergic Neurons in Nucleus Raphe Magnus in Opioid Antinociception Martin Wessendorf
It has been proposed that serotonergic neurons do not mediate opioid analgesia, based on the observation that the firing rates of serotonergic neurons are not modulated by antinociceptive treatments such as systemic opioidsor periaqueductal gray (PAG) stimulation. However, several points need to be clarified regarding the data supporting this hypothesis. These include establishing whether anesthesia has influenced the results of these experiments, whether the serotonergic neurons recorded project to the dorsal horn, and whether presynaptic effects might allow opioids to change serotonin releasewithout changing neuronal firing rate. Key words: 5-hydroxytryptamine (5-HT), morphine, opiate, periaqueductal gray.
ason and Gao present a hypothesis that attempts to resolve an apparent contradiction between data supporting a role for serotonergic mediation of opiate antinociception on the one hand and their own data suggesting that serotonergic neurons do not change their firing in response to antinociceptive treatments. Their data are both impressive and intriguing, and Mason and Gao may be correct: descending serotonergic systems may have less to do with mediating analgesia than has been proposed in the past. However, although their data raise some fundamental questions, they also have some shortcomings. In their paper on periaqueductal gray (PAG) stimulation [9], Gao and Mason and their co-workers reported
M
From the Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN. Reprint requests: Martin Wessendorf, PhD., Department of Cell Biology and Neuroanatomy, University of Minnesota, 321 Church St. SE, Minneapolis, MN 55455. ©
1998 the American Pain Society
1058-9139/0703-0010$5.00/0
Pain Forum 7(3): 159-162, 1998
that brief shocks to the PAG were capable of exciting neurons within nucleus raphe magnus (NRM). They found that the population of cells that were excited did not appear to include the serotonergic cells that they have identified. Moreover, more prolonged trains of stimuli-trains that resulted in prolongation of the tailflick latency-similarly appeared not to affect the firing rate of the serotonergic cells they record. In both this and a later paper (see below), their conclusions are based on their ability to identify serotonergic neurons. They have done so by intracellularly filling neurons and staining them for serotonin. Post-hoc analysis of their data has allowed them to conclude that a neuron is serotonergic if, and only if, it has a slow, regular firing rate. This criterion was based on their observations that virtually all cells with slow, regular firing rates were labeled for serotonin, and that few or no cells that the authors sampled were immunoreactive for serotonin if they had firing patterns that were not slow and regular. Mason and Gao concluded from this study that few or no serotonergic neurons in NRM are excited by PAG stimulation. Gao et al. [9] acknowledge reports that serotonergic antagonists decrease antinociceptive and cardiovascular responses to PAG stimulation but argue that antagonizing serotonin will decrease those responses, whether or not PAG stimulation evokes increased release. In a second paper, Gao et al. examined the responses to morphine of serotonergic neurons in NRM [10]. It was found that just under one-fourth of serotonergic neurons responded to a dose of 1 mg/kg of morphine. Moreover, when higher doses were used, responses were observed in almost two-thirds of the serotonergic cells recorded (Fig. 4 in [10]); the proportion of responding cells was significantly higher than at 1 mg/kg, suggesting a dose-related effect. At both doses, the responses were split approximately evenly between excitation and inhibition, and in all but one case, the doses administered
159
160
COMMENTARYlWessendorf
were sufficient to suppress the tail-withdrawal reflex. However, the responses were reported to be inconsistent and appeared to the authors to correlate better with the effects of morphine on heart rate. As with the first study, the investigators conclude that morphine antinociception is not mediated through excitation of serotonergic neurons . However, given that there is reasonably good evidence that activation of 5-hydroxytryptamine (5-HT) receptors alters nociception [9,10], the investigators argue that serotonergic neurons must exert a tonic effect on nociception, rather than a phasic effect. The investigators' hypothesis provides an interesting and valuable point of departure for discussing a frequently confusing body of literature. My criticisms are largely that their data may not be as strong as they suggest , and that there may be alternate explanations for their observations. First, they state that serotonergic neurons are neither necessary nor sufficient for the antinociceptive effects of PAG stimulation or systemic morphine. Their data support their contention that serotonergic neurons are not necessary for these phenomena. However, they appear not to have tested whether 5-HT neurons are sufficient to support antinociception . It appears likely that, in fact, NRM serotonergic neurons at least partially mediate some opioid effects. It has been reported that microinjection of opioids into the PAG results in release of 5-HT in the spinal cord [29]. Furthermore, it has been reported that when opioids are microinjected into the PAG, their antinociceptive effects can be antagonized by microinjection of «-opiold agonists into NRM. This effect appears to be mediated by direct inhibition of an electrophysiologically distinct category of neuron referred to as a primary cell [16]. Previous studies have shown that the great majority of primary cells are immunoreactive for serotonin [17]. Thus, it appears likely that serotonergic neurons mediate some or all of the effects of opioid application into the PAG. Although the investigators argue that NRM serotonergic neurons do not mediate the antinociceptive effects of electrical stimulation of the PAG, they have not directly determined the effects of PAG morphine . Second, the investigators appear not to have considered the possibility that their use of an anesthetized preparation has altered the results of their studies. It has been reported that anesthesia suppresses the effects of a variety of physiological stimuli on serotonergic neurons [21]. Given those findings, it appears,possible that the lack of change in firing rate that they observe is due to anesthesia. Ideafly, the experiments could be repeated in an unanesthetized animal. However, it is critical that the neurons recorded be confirmed to be serotonergic. The criterion that the investigators developed to identify serotonergic neurons in the anesthetized preparation was valuable because it was verified by combining
immunocytochemistry with intracellular filling of cells , and, given the effects of anesthesia on serotonergic cells [21], this characterization would need to be repeated in awake animals. Third , as the investigators have acknowledged [9], it is unclear whether they have sampled the relevant population of serotonergic NRM neurons . 5-HT neurons in the caudal pons and medulla are organized by their projections and there exist at least two distinct populations of 5-HT cells. Projecting to the ventral horn and intermediate gray are serotonergic neurons that express the neuropeptides substance P and/or thyrotropin-releasing hormone (TRH) [1,4,20,22,25]. Projecting to the superficial dorsal horn are serotonergic neurons that express neither substance P nor TRH [1,4,22,25]. Both peptidecontaining and non-peptide-containing serotonergic cells have been reported to be common in NRM [2,11,12]. However, the anatomic segregation of the projections of these two types of serotonergic neurons strongly suggests that there are also functional differences between them. Moreover, there is pharmacologic evidence for there being two electrophysiologically distinct categories of serotonergic NRM neurons. Almost all NRM neurons with conduction velocities slower than 6 m/sec appear to be sensitive to the serotonin neurotoxin 5,7-dihydroxytryptamine, which suggests that these cells are serotonergic [26]. This range of conduction velocities is consistent with ultrastructural studies of 5-HT axon diameters [5,19,27]. Of those neurons, the cells with conduction velocities slower than 1.2 m/sec were reported to have slow, regular firing rates similar to those recorded by Mason and Gao. In contrast , those cells conducting between 2 and 6 m/sec had no consistent pattern of spontaneous discharge [24] and therefore would not have been classified as serotonergic by the criterion of Mason [14]. Thus, it appears likely that their sample included only one of the major functional groups of serotonergic neurons. In light of these considerations, it remains unclear whether the serotonergic neurons recorded by Mason and Gao project to the spinal dorsal horn. Antidromic tracking of axonal projections might be a means of resolving this issue [7]. Fourth, there - is evidence that presynaptic effects might allow opioids to affect 5-HT release without affecting firing rate. My co-workers and I have reported that immunoreactivity for the cloned ()-opioid receptor (DOR1) occurs on serotonergic varicosities in the spinal cord [3], and the expression of DOR1 by bulbospinal 5-HT cells has subsequently been confirmed using in situ hybridization [23]. The expression of DOR1-ir by 5-HT-ir processes was reported in the spinal ventral horn, but was also found in nucleus proprius of the dorsal horn [3]. The
COMMENTARYlWessendorf
evidence for a presynaptic action of opioids on spinal 5-HT release has been equivocal, sometimes requiring relatively high doses to observe results [15]. However, a recent study has reported that relatively low doses of o-opioid agonists inhibit the release of 5-HT from ventral horn preparations [8]. It appears likely that a similar effect occurs among the corresponding serotonergic fibers in the neck of the dorsal horn. The most probable result of decreased 5-HT release would appear to be an increase in nociception [28], but it is also possible that it would result in antinociception depending upon the specifics of the circuitry and receptors involved. Can any single set of experiments resolve all the contradictions that exist in this field? I expect not. For instance, my co-workers and I recently reported that NRM serotonergic neurons retrogradely labeled from the dorsal spinal cord express immunoreactivity for the cloned p-opioid receptor [13]. More recently, we have confirmed the presence of this receptor using in situ hybridization [23]. Gao et al. [10] reported that about one-third of serotonergic neurons were inhibited by doses of morphine higher than 1 mg/kg, which would appear to be consistent with our findings. However, the evidence to date is that activation of u-oploid receptors has no direct effects on serotonergic NRM neuronsonly indirect effects [17,18]. Therefore, other factors, perhaps including genetic differences between rats from different vendors [6] and electrode bias, may also need to be considered as possible sources of contradictory data. Thus, although I share the investigators' distrust of the present dogma, I do not think that we yet know the role of serotonergic neurons in opioid antinociception. The questions are difficult; the jury is still out.
References 1. Arvidsson U, Cullheim S, Ulfhake Bet al: 5-Hydroxytryptamine, substance P, and thyrotropin-releasing hormone in the adult cat spinal cord segment L7: immunohistochemical and chemical studies. Synapse 6:237-270, 1990 2. Arvidsson U, Cullheim S, Ulfhake B et al: Quantitative and qualitative aspects on the distribution of 5-HT and its coexistence with substance P and TRH in cat ventral medullary neurons [published erratum appears in J Chem Neuroanat 1994 Oct;7(4):285-287]. J Chem Neuroanat 7:3-12,1994 3. Arvidsson U, Dado RJ, Riedl M et al: 8-0pioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J Neurosci 15:1215-1235, 1995 4. Arvidsson U, Ulfhake B, Cullheim S et al: Thyrotropinreleasing hormone (TRH)-like immunoreactivity in the grey monkey (Macaca fascicularis) spinal cord and medulla
161
oblongata with special emphasis on the bulbospinal tract. J Comp Neurol 322:293-310, 1992 5. Basbaum AI, Zahs K, Lord B, Lakos S: The fiber caliber of 5-HT immunoreactive axons in the dorsolateral funiculus of the spinal cord of the rat and cat. Somatosens Res 5:177-185,1988 6. Clark FM, Proudfit HK: Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons. Brain Res 591:4453, 1992 7. Fields HL, Malick A, Burstein R: Dorsal horn projection targets of ON and OFF cells in the rostral ventromedial medulla. J NeurophysioI74:1742-1759, 1995 8. Franck J, Nylander I, Rosen A: Met-enkephalin inhibits 5-hydroxytryptamine release from the rat ventral spinal cord via delta opioid receptors. Neuropharmacology 35: 743-749,1996 9. Gao K, Kim YH, Mason P: SEROTONERGIC pontomedullary neurons are not activated by antinociceptive stimulation in the periaqueductal gray. J Neurosci 17:3285-3292, 1997 10. Gao KM, Chen DO, Genzen JR, Mason P: Activation of serotonergic neurons in the raphe magnus is not necessary for morphine analgesia. Neurosci 18:1860-1868, 1998 11. Johansson 0, HokfeltT, Pernow B et al: Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord. Neuroscience 6:1857-1881, 1981 12. Kachidian, P, Poulat P, Marlier L, Privat A: Immunohistochemical evidence for the coexistence of substance P, thyrotropin-releasing hormone, GABA, methionine-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: a dual labeling in the rat. J Neurosci Res 30:521-530,1991 13. Kalyuzhny AE, Arvidsson U, Wu W, Wessendorf MW: mu-Opioid and delta-opioid receptors are expressed in brainstem antinociceptive circuits: studies using immunocytochemistry and retrograde tract-tracing. J Neurosci 16:6490-6503, 1996 14. Mason P: Physiological identification of pontomedullary serotonergic neurons in the rat. J Neurophysiol 77:10871098, 1997 15. Monroe PJ, Kradel BK, Smith DL, Smith OJ: Opioid effects on spinal [3H]5-hydroxytryptamine release are not related to their antinociceptive action. Eur J Pharmacol 272:5156,1995 16. Pan ZZ, Tershner SA, Fields HL: Cellular mechanism for anti-analgesic action of agonists of the kappa-opioid receptor. Nature 389:382-385, 1997 17. Pan ZZ, Wessendorf MW, Williams JT: Modulation by serotonin of the neurons in rat nucleus raphe magnus in vitro. Neuroscience 54:421-429, 1993
162
COMMENTARY/Wessendorf
18. Pan ZZ, Williams JT, Osborne PB: Opioid actions on single nucleus raphe magnus neurons from rat and guinea-pig in vitro. J Physiol (Lond) 427:519-532,1990 19. Ruda MA, and Gobel S: Ultrastructural characterization of axonal endings in the substantia gelatinosa which take up [3H]serotonin. Brain Res 184:57-83, 1980 20. Sasek CA, Wessendorf MW, Helke CJ: Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neuroscience 35:105-119, 1990 21. Tao R, Auerbach SB: Anesthetics block morphine-induced increases in serotonin release in rat CNS. Synapse 18:307314~ 1994 22. Tashiro T, Ruda MA: Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord. Peptides 9:383-391, 1988 23. Wang H, Law P, Wessendorf MW: Mu- and delta-opioid receptor mRNAs are expressed in spinally projecting RVM neurons. Soc Neurosci Abstr 23:444, 1997
24. Wessendorf, MW and Anderson, EG: Single unit studies of identified bulbospinal serotonergic units. Brain Res 279:93103,1983 25. Wessendorf MW, Elde R: The coexistence of serotoninand substance P-like immunoreactivity in the spinal cord of the rat as shown by immunofluorescent double labeling. J Neurosci 7:2352-2363, 1987 26. Wessendorf MW, Proudfit HK, Anderson EG: The identification of serotonergic neurons in the nucleus raphe magnus by conduction velocity. Brain Res 214:168-173, 1981 27. Westlund KN, Lu Y, Coggeshall RE, Willis WD: Serotonin is found in myelinated axons of the dorsolateral funiculus in monkeys. Neurosci Lett 141:35-38, 1992 28. Willcockson WS, Chung JM, Hori Y et al: Effects of iontophoretically released amino acids and amines on primate spinothalamic tract cells. J Neurosci 4:732-740, 1984 29. Yaksh TL, and Tyee GM: Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from spinal cord. Brain Res 171:176-181,1979
The Role of Serotonergic Neurons in Nucleus Raphe Magnus in Opioid Antinociception Martin Wessendorf
It has been proposed that serotonergic neurons do not mediate opioid analgesia, based on the observation that the firing rates of serotonergic neurons are not modulated by antinociceptive treatments such as systemic opioidsor periaqueductal gray (PAG) stimulation. However, several points need to be clarified regarding the data supporting this hypothesis. These include establishing whether anesthesia has influenced the results of these experiments, whether the serotonergic neurons recorded project to the dorsal horn, and whether presynaptic effects might allow opioids to change serotonin releasewithout changing neuronal firing rate. Key words: 5-hydroxytryptamine (5-HT), morphine, opiate, periaqueductal gray.
ason and Gao present a hypothesis that attempts to resolve an apparent contradiction between data supporting a role for serotonergic mediation of opiate antinociception on the one hand and their own data suggesting that serotonergic neurons do not change their firing in response to antinociceptive treatments. Their data are both impressive and intriguing, and Mason and Gao may be correct: descending serotonergic systems may have less to do with mediating analgesia than has been proposed in the past. However, although their data raise some fundamental questions, they also have some shortcomings. In their paper on periaqueductal gray (PAG) stimulation [9], Gao and Mason and their co-workers reported
M
From the Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN. Reprint requests: Martin Wessendorf, PhD., Department of Cell Biology and Neuroanatomy, University of Minnesota, 321 Church St. SE, Minneapolis, MN 55455. ©
1998 the American Pain Society
1058-9139/0703-0010$5.00/0
Pain Forum 7(3): 159-162, 1998
that brief shocks to the PAG were capable of exciting neurons within nucleus raphe magnus (NRM). They found that the population of cells that were excited did not appear to include the serotonergic cells that they have identified. Moreover, more prolonged trains of stimuli-trains that resulted in prolongation of the tailflick latency-similarly appeared not to affect the firing rate of the serotonergic cells they record. In both this and a later paper (see below), their conclusions are based on their ability to identify serotonergic neurons. They have done so by intracellularly filling neurons and staining them for serotonin. Post-hoc analysis of their data has allowed them to conclude that a neuron is serotonergic if, and only if, it has a slow, regular firing rate. This criterion was based on their observations that virtually all cells with slow, regular firing rates were labeled for serotonin, and that few or no cells that the authors sampled were immunoreactive for serotonin if they had firing patterns that were not slow and regular. Mason and Gao concluded from this study that few or no serotonergic neurons in NRM are excited by PAG stimulation. Gao et al. [9] acknowledge reports that serotonergic antagonists decrease antinociceptive and cardiovascular responses to PAG stimulation but argue that antagonizing serotonin will decrease those responses, whether or not PAG stimulation evokes increased release. In a second paper, Gao et al. examined the responses to morphine of serotonergic neurons in NRM [10]. It was found that just under one-fourth of serotonergic neurons responded to a dose of 1 mg/kg of morphine. Moreover, when higher doses were used, responses were observed in almost two-thirds of the serotonergic cells recorded (Fig. 4 in [10]); the proportion of responding cells was significantly higher than at 1 mg/kg, suggesting a dose-related effect. At both doses, the responses were split approximately evenly between excitation and inhibition, and in all but one case, the doses administered
159
160
COMMENTARYlWessendorf
were sufficient to suppress the tail-withdrawal reflex. However, the responses were reported to be inconsistent and appeared to the authors to correlate better with the effects of morphine on heart rate. As with the first study, the investigators conclude that morphine antinociception is not mediated through excitation of serotonergic neurons . However, given that there is reasonably good evidence that activation of 5-hydroxytryptamine (5-HT) receptors alters nociception [9,10], the investigators argue that serotonergic neurons must exert a tonic effect on nociception, rather than a phasic effect. The investigators' hypothesis provides an interesting and valuable point of departure for discussing a frequently confusing body of literature. My criticisms are largely that their data may not be as strong as they suggest , and that there may be alternate explanations for their observations. First, they state that serotonergic neurons are neither necessary nor sufficient for the antinociceptive effects of PAG stimulation or systemic morphine. Their data support their contention that serotonergic neurons are not necessary for these phenomena. However, they appear not to have tested whether 5-HT neurons are sufficient to support antinociception . It appears likely that, in fact, NRM serotonergic neurons at least partially mediate some opioid effects. It has been reported that microinjection of opioids into the PAG results in release of 5-HT in the spinal cord [29]. Furthermore, it has been reported that when opioids are microinjected into the PAG, their antinociceptive effects can be antagonized by microinjection of «-opiold agonists into NRM. This effect appears to be mediated by direct inhibition of an electrophysiologically distinct category of neuron referred to as a primary cell [16]. Previous studies have shown that the great majority of primary cells are immunoreactive for serotonin [17]. Thus, it appears likely that serotonergic neurons mediate some or all of the effects of opioid application into the PAG. Although the investigators argue that NRM serotonergic neurons do not mediate the antinociceptive effects of electrical stimulation of the PAG, they have not directly determined the effects of PAG morphine . Second, the investigators appear not to have considered the possibility that their use of an anesthetized preparation has altered the results of their studies. It has been reported that anesthesia suppresses the effects of a variety of physiological stimuli on serotonergic neurons [21]. Given those findings, it appears,possible that the lack of change in firing rate that they observe is due to anesthesia. Ideafly, the experiments could be repeated in an unanesthetized animal. However, it is critical that the neurons recorded be confirmed to be serotonergic. The criterion that the investigators developed to identify serotonergic neurons in the anesthetized preparation was valuable because it was verified by combining
immunocytochemistry with intracellular filling of cells , and, given the effects of anesthesia on serotonergic cells [21], this characterization would need to be repeated in awake animals. Third , as the investigators have acknowledged [9], it is unclear whether they have sampled the relevant population of serotonergic NRM neurons . 5-HT neurons in the caudal pons and medulla are organized by their projections and there exist at least two distinct populations of 5-HT cells. Projecting to the ventral horn and intermediate gray are serotonergic neurons that express the neuropeptides substance P and/or thyrotropin-releasing hormone (TRH) [1,4,20,22,25]. Projecting to the superficial dorsal horn are serotonergic neurons that express neither substance P nor TRH [1,4,22,25]. Both peptidecontaining and non-peptide-containing serotonergic cells have been reported to be common in NRM [2,11,12]. However, the anatomic segregation of the projections of these two types of serotonergic neurons strongly suggests that there are also functional differences between them. Moreover, there is pharmacologic evidence for there being two electrophysiologically distinct categories of serotonergic NRM neurons. Almost all NRM neurons with conduction velocities slower than 6 m/sec appear to be sensitive to the serotonin neurotoxin 5,7-dihydroxytryptamine, which suggests that these cells are serotonergic [26]. This range of conduction velocities is consistent with ultrastructural studies of 5-HT axon diameters [5,19,27]. Of those neurons, the cells with conduction velocities slower than 1.2 m/sec were reported to have slow, regular firing rates similar to those recorded by Mason and Gao. In contrast , those cells conducting between 2 and 6 m/sec had no consistent pattern of spontaneous discharge [24] and therefore would not have been classified as serotonergic by the criterion of Mason [14]. Thus, it appears likely that their sample included only one of the major functional groups of serotonergic neurons. In light of these considerations, it remains unclear whether the serotonergic neurons recorded by Mason and Gao project to the spinal dorsal horn. Antidromic tracking of axonal projections might be a means of resolving this issue [7]. Fourth, there - is evidence that presynaptic effects might allow opioids to affect 5-HT release without affecting firing rate. My co-workers and I have reported that immunoreactivity for the cloned ()-opioid receptor (DOR1) occurs on serotonergic varicosities in the spinal cord [3], and the expression of DOR1 by bulbospinal 5-HT cells has subsequently been confirmed using in situ hybridization [23]. The expression of DOR1-ir by 5-HT-ir processes was reported in the spinal ventral horn, but was also found in nucleus proprius of the dorsal horn [3]. The
COMMENTARYlWessendorf
evidence for a presynaptic action of opioids on spinal 5-HT release has been equivocal, sometimes requiring relatively high doses to observe results [15]. However, a recent study has reported that relatively low doses of o-opioid agonists inhibit the release of 5-HT from ventral horn preparations [8]. It appears likely that a similar effect occurs among the corresponding serotonergic fibers in the neck of the dorsal horn. The most probable result of decreased 5-HT release would appear to be an increase in nociception [28], but it is also possible that it would result in antinociception depending upon the specifics of the circuitry and receptors involved. Can any single set of experiments resolve all the contradictions that exist in this field? I expect not. For instance, my co-workers and I recently reported that NRM serotonergic neurons retrogradely labeled from the dorsal spinal cord express immunoreactivity for the cloned p-opioid receptor [13]. More recently, we have confirmed the presence of this receptor using in situ hybridization [23]. Gao et al. [10] reported that about one-third of serotonergic neurons were inhibited by doses of morphine higher than 1 mg/kg, which would appear to be consistent with our findings. However, the evidence to date is that activation of u-oploid receptors has no direct effects on serotonergic NRM neuronsonly indirect effects [17,18]. Therefore, other factors, perhaps including genetic differences between rats from different vendors [6] and electrode bias, may also need to be considered as possible sources of contradictory data. Thus, although I share the investigators' distrust of the present dogma, I do not think that we yet know the role of serotonergic neurons in opioid antinociception. The questions are difficult; the jury is still out.
References 1. Arvidsson U, Cullheim S, Ulfhake Bet al: 5-Hydroxytryptamine, substance P, and thyrotropin-releasing hormone in the adult cat spinal cord segment L7: immunohistochemical and chemical studies. Synapse 6:237-270, 1990 2. Arvidsson U, Cullheim S, Ulfhake B et al: Quantitative and qualitative aspects on the distribution of 5-HT and its coexistence with substance P and TRH in cat ventral medullary neurons [published erratum appears in J Chem Neuroanat 1994 Oct;7(4):285-287]. J Chem Neuroanat 7:3-12,1994 3. Arvidsson U, Dado RJ, Riedl M et al: 8-0pioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J Neurosci 15:1215-1235, 1995 4. Arvidsson U, Ulfhake B, Cullheim S et al: Thyrotropinreleasing hormone (TRH)-like immunoreactivity in the grey monkey (Macaca fascicularis) spinal cord and medulla
161
oblongata with special emphasis on the bulbospinal tract. J Comp Neurol 322:293-310, 1992 5. Basbaum AI, Zahs K, Lord B, Lakos S: The fiber caliber of 5-HT immunoreactive axons in the dorsolateral funiculus of the spinal cord of the rat and cat. Somatosens Res 5:177-185,1988 6. Clark FM, Proudfit HK: Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons. Brain Res 591:4453, 1992 7. Fields HL, Malick A, Burstein R: Dorsal horn projection targets of ON and OFF cells in the rostral ventromedial medulla. J NeurophysioI74:1742-1759, 1995 8. Franck J, Nylander I, Rosen A: Met-enkephalin inhibits 5-hydroxytryptamine release from the rat ventral spinal cord via delta opioid receptors. Neuropharmacology 35: 743-749,1996 9. Gao K, Kim YH, Mason P: SEROTONERGIC pontomedullary neurons are not activated by antinociceptive stimulation in the periaqueductal gray. J Neurosci 17:3285-3292, 1997 10. Gao KM, Chen DO, Genzen JR, Mason P: Activation of serotonergic neurons in the raphe magnus is not necessary for morphine analgesia. Neurosci 18:1860-1868, 1998 11. Johansson 0, HokfeltT, Pernow B et al: Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord. Neuroscience 6:1857-1881, 1981 12. Kachidian, P, Poulat P, Marlier L, Privat A: Immunohistochemical evidence for the coexistence of substance P, thyrotropin-releasing hormone, GABA, methionine-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: a dual labeling in the rat. J Neurosci Res 30:521-530,1991 13. Kalyuzhny AE, Arvidsson U, Wu W, Wessendorf MW: mu-Opioid and delta-opioid receptors are expressed in brainstem antinociceptive circuits: studies using immunocytochemistry and retrograde tract-tracing. J Neurosci 16:6490-6503, 1996 14. Mason P: Physiological identification of pontomedullary serotonergic neurons in the rat. J Neurophysiol 77:10871098, 1997 15. Monroe PJ, Kradel BK, Smith DL, Smith OJ: Opioid effects on spinal [3H]5-hydroxytryptamine release are not related to their antinociceptive action. Eur J Pharmacol 272:5156,1995 16. Pan ZZ, Tershner SA, Fields HL: Cellular mechanism for anti-analgesic action of agonists of the kappa-opioid receptor. Nature 389:382-385, 1997 17. Pan ZZ, Wessendorf MW, Williams JT: Modulation by serotonin of the neurons in rat nucleus raphe magnus in vitro. Neuroscience 54:421-429, 1993
162
COMMENTARY/Wessendorf
18. Pan ZZ, Williams JT, Osborne PB: Opioid actions on single nucleus raphe magnus neurons from rat and guinea-pig in vitro. J Physiol (Lond) 427:519-532,1990 19. Ruda MA, and Gobel S: Ultrastructural characterization of axonal endings in the substantia gelatinosa which take up [3H]serotonin. Brain Res 184:57-83, 1980 20. Sasek CA, Wessendorf MW, Helke CJ: Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neuroscience 35:105-119, 1990 21. Tao R, Auerbach SB: Anesthetics block morphine-induced increases in serotonin release in rat CNS. Synapse 18:307314~ 1994 22. Tashiro T, Ruda MA: Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord. Peptides 9:383-391, 1988 23. Wang H, Law P, Wessendorf MW: Mu- and delta-opioid receptor mRNAs are expressed in spinally projecting RVM neurons. Soc Neurosci Abstr 23:444, 1997
24. Wessendorf, MW and Anderson, EG: Single unit studies of identified bulbospinal serotonergic units. Brain Res 279:93103,1983 25. Wessendorf MW, Elde R: The coexistence of serotoninand substance P-like immunoreactivity in the spinal cord of the rat as shown by immunofluorescent double labeling. J Neurosci 7:2352-2363, 1987 26. Wessendorf MW, Proudfit HK, Anderson EG: The identification of serotonergic neurons in the nucleus raphe magnus by conduction velocity. Brain Res 214:168-173, 1981 27. Westlund KN, Lu Y, Coggeshall RE, Willis WD: Serotonin is found in myelinated axons of the dorsolateral funiculus in monkeys. Neurosci Lett 141:35-38, 1992 28. Willcockson WS, Chung JM, Hori Y et al: Effects of iontophoretically released amino acids and amines on primate spinothalamic tract cells. J Neurosci 4:732-740, 1984 29. Yaksh TL, and Tyee GM: Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from spinal cord. Brain Res 171:176-181,1979