Activation of serotonergic neurons in the raphe magnus is not necessary for morphine analgesia

Abstract

A wealth of pharmacological and behavioral data suggests that spinally projecting serotonergic cells mediate opioid analgesia. A population of medullary neurons, located within raphe magnus (RM) and the neighboring reticular nuclei, contains serotonin and is the source of serotonin in the spinal dorsal horn. To test whether serotonergic neurons mediate opioid analgesia, morphine was administered during recordings from medullary cells that were physiologically characterized as serotonergic (5HTp) by their slow and steady discharge pattern in the lightly anesthetized rat. Selected 5HTp cells (n = 14) were intracellularly labeled, and all contained serotonin immunoreactivity. The discharge of most 5HTp cells was not affected by an analgesic dose of systemic morphine. In a minority of cases, 5HTp cells either increased or decreased their discharge after morphine administration. However, morphine altered the discharge of some 5HTp cells in the absence of producing analgesia and conversely did not alter the discharge of most 5HTp cells in cases in which analgesia occurred. RM cells with irregular discharge patterns and excitatory or inhibitory responses to noxious tail heat were classified as ON and OFF cells, respectively. All ON and OFF cells that were intracellularly labeled (n = 9) lacked serotonin immunoreactivity. All ON cells were inhibited, and most OFF cells were excited by systemic morphine. Because 5HTp cells do not consistently change their discharge during morphine analgesia, they are unlikely to mediate the analgesic effects of morphine. Instead, nonserotonergic cells are likely to mediate morphine analgesia in the anesthetized rat. In light of the sensitivity of morphine analgesia to manipulations of serotonin, serotonin release, although neither necessary nor sufficient for opioid analgesia, is proposed to facilitate the analgesic effects of nonserotonergic RM terminals in the spinal cord.

Fig. 1.

Recording sites on nissl-stained coronal sections of the ventromedial medulla. Recording sites for 5HT_p(○), 5HT_p/ir (•), on cells (upward triangles), and off cells (downward triangles). 5HT_p cells were identified by physiological criteria alone, whereas 5HT_p/ir cells were initially identified physiologically and then were labeled and found to contain serotonin immunoreactivity. on and offcells that were immunochemically confirmed to be nonserotonergic are shown as filled symbols. The number below each section is the location of that section relative to interaural zero (in millimeters).

Fig. 2.

Physiological characteristics of recorded cells. The coefficient of variation of the interspike interval (CV_ISI) is plotted against the mean interspike interval for a 5 min period of background discharge. Aline representing the optimal linear boundary between serotonergic and nonserotonergic cells is illustrated on this same graph. 5HT_p (○); 5HTp/ir (•); on cells (upward triangles); and off cells (downward triangles). On and offcells that were immunochemically confirmed to be nonserotonergic are shown as filled symbols.

Fig. 3.

Serotonin immunoreactivity in intracellularly labeled cells. The intracellular label visualized with Texas Red (A1–F1) and serotonin immunoreactivity visualized with Bodipy (A2–F2) are shown for serotonergic (A–D) and off (E, F) cells.

Fig. 4.

Effect of morphine on 5HT_p cell discharge. The average discharge rate for each 5HT_p cell, during the 60 sec before each tail heat stimulation, is shown for baseline, morphine, and naloxone conditions. Tail flick withdrawals occurred at time points marked with a filled circle and were suppressed at time points marked with an open circle. A, 5HT_p cells whose discharge was unaffected by administration of ≤1 mg/kg morphine.B, 5HT_p cells whose discharge was affected by administration of 1 mg/kg morphine. C, 5HT_p cells whose discharge was unaffected by administration of >1 mg/kg morphine. D, 5HT_p cells whose discharge was affected by administration of >1 mg/kg morphine.

Fig. 5.

Representative recordings from RM and NRMC serotonergic cells before and after morphine administration. The traces are labeled in C and are (top tobottom) heart rate, mean arterial blood pressure, neuronal discharge rate, rectified paraspinous EMG, and thermal tail stimulus. The scales for the neuronal discharge and heart rate (in bpm) are on the left, and the scale for blood pressure (in mm Hg) is on the right. Injections of morphine and naloxone were administered at times indicated by the labeled arrows below the tail stimulus trace. A, Continuous record from a 5HT_p/ir cell that was unaffected by 1.0 mg/kg morphine and 1 mg/kg naloxone. B, Continuous record from a 5HT_p cell that transiently increased its discharge after 2 mg/kg morphine and was unaffected by 1 mg/kg naloxone.C, Continuous record from a 5HT_p/ir cell that transiently decreased its discharge after 1 mg/kg morphine and was unaffected by 1 mg/kg naloxone.

Fig. 6.

Evoked responses from recorded cells before and after morphine and naloxone. The bottom trace represents instantaneous discharge rate of the cell. The middle trace represents mean arterial blood pressure, and thetop trace shows the instantaneous heart rate. The scale bar for the neuronal discharge rate (in Hz) is on theleft. The small scale on the right is for blood pressure (0–100 mmHg), and the large scale on theright (bpm) is for heart rate. The barsbelow the unit trace indicate the application of noxious tail heat, and the arrows indicate the time of the withdrawal. In cases in which the animal did not withdraw, there is no arrow. Baseline, post-morphine, and post-naloxone responses are shown in theleft, middle, and right columns, respectively. Each trace is 100 sec in duration. A, A 5HT_p cell that was unresponsive to noxious heat.B, A 5HT_p/ir cell that was excited by noxious heat. C, An off cell.D, An on cell.