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Release of endogenous monoamines into spinal cord superfusates following the microinjection of phentolamine into the nucleus raphe magnus
246
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Release of endogenous monoamines into spinal cord superfusates following the microinjection of phentolamine into the nucleus raphe magnus Jacqueline Sagen and Herbert K. Proudfit Department of Pharmacology, University of lllinois College of Medicine at Chicago, Chicago, 1L 60680 ( U.S.A ) (Accepted 12 August 1986) Key words: Antinociception; Nucleus raphe magnus; Norepinephrine; Serotonin; Spinal cord; Superfusion; Neurotransmitter release
Previous studies have suggested that raphe-spinal neurons located in the nucleus raphe magnus (NRM) are tonically inhibited by noradrenergic neurons. Furthermore, blockade of the inhibitory noradrenergic input to the NRM induces antinociception which appears to be mediated by the release of both serotonin and norepinephrine in the spinal cord. The present experiments were designed to directly measure the release of endogenous serotonin and norepinephrine into spinal cord superfusates before and after the microinjection of the a-adrenergic antagonist phentolamine into the NRM. High-performance liquid chromatography with electrochemical detection was used to quantitate the monoamines. The injection of phentolamine into the NRM induced a significant increase in the amount of both norepinephrine and serotonin released in the spinal cord. This enhanced release was not observed following either the injection of phentolamine into sites outside the NRM or the injection of saline vehicle into the NRM. These results support the proposal that the antinociception induced by the blockade of the inhibitory noradrenergic input to the NRM is mediated by the activation of spinally-projecting serotonergic and noradrenergic neurons.
INTRODUCTION It is well established that neurons in the nucleus raphe magnus (NRM) play an important role in modulating the perception of noxious stimuli 3'2°'54'55. The activity of these N R M neurons appears to be under the tonic inhibitory control of brainstem N A neurons 51,53. Blockade of this inhibitory N A input by the microinjection of a-adrenergic antagonists induces potent antinociception 21'22'41'43'45. Previous studies have suggested that this antinociception is mediated by spinally-projecting monoaminergic neurons, since it is reversed by the injection of either serotonergic 22 or a-adrenergic 41 antagonists into the spinal cord subarachnoid space. Furthermore, the depletion of spinal cord serotonin (5-HT) and/or norepinephrine (NE) content by neurotoxins severely attenuates or blocks the induction of such antinociception 45.
Together, these studies support the proposal that disinhibition of N R M neurons produced by the blockade of N A receptors in this region inhibits nociceptive transmission in the spinal cord by: (1) activation of raphe-spinal serotenergic neurons; and (2) activation of bulbospinal N A neurons. This implies that such antinociception results from the release of both 5-HT and N E from axon terminals in the spinal cord. Recent studies have demonstrated that it is possible to measure changes in the concentration of these monoamines in spinal cord superfusates following pharmacological and physiological manipulations 24,49,56,57. The purpose of this study was to directly measure the release of endogenous N E and 5-HT in the spinal cord following the injection of the a-adrenergic antagonist phentolamine into the NRM. A preliminary account of these experiments has been reported 39.
Correspondence: H.K. Proudfit, Department of Pharmacology, University of IUinois College of Medicine at Chicago, P.O. Box 6998, Chicago, IL 60680, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
247 MATERIALSAND METHODS
Surgicalprocedures Sprague-Dawley derived rats weighing 350-400 g were surgically prepared for drug microinjection and spinal cord superfusion. A chronic indwelling stainless steel microinjection guide tube (23-gauge) aimed at the NRM (P 2.5 mm; H - 0 . 5 mm; L 0.00 mm; incisor bar -2.5 mm) was implanted at least one week prior to superfusion procedures (for details see Sagen and Proudfit43). Animals that exhibited any obvious behavioral or motor abnormalities following cannula insertion were not used. The remaining animals were housed individually and allowed free access to food and water.
Microin]ectionprocedures Drug solutions were microinjected through a 30gauge stainless steel injection cannula inserted into the indwelling guide tube. The tip of the injection cannula extended 4 mm beyond the end of the guide tube to minimize damage to the injection site. The injection cannula was connected to an electrical geardriven syringe pump (Harvard Apparatus) by a length of tubing (PE 20) filled with drug solution. The volume of each injection was 0.5/~1 delivered over a period of 60 s and monitored by observing the movement of an air bubble over a calibrated distance in the tubing. The injection cannula was left in place for 60 s after the injection to minimize flow of the drug solution up the cannula track.
Spinal cord superfusion procedures Spinal cord superfusion procedures were performed 1 week following cannula insertion. Animals were anesthetized with urethane (1200 mg/kg), fitted with a tracheal tube and immobilized in a stereotaxic head holder. An intrathecal catheter made of PE 10 tubing was threaded down the intrathecal space through a small slit in the atlanto-occipital membrane to the rostral tip of the lumbar enlargement. The catheter was connected to a 20-ml glass syringe containing artificial cerebrospinal fluid and served to infuse fluid. The outflow from the spinal cord was collected using a length of polyethylene tubing (PE 50) which was flared at the end and placed over the opening in the dura. This tubing was connected to a 3-way stopcock so that the outflow could be directed to one
of two collection tubes (50 mm long glass tubing, 3 mm internal diameter) which were kept cold on an ice slurry. These tubes were connected to a second 20-ml glass syringe. This arrangement allowed sequential samples to be collected without stopping the flow of the superfusion. The infusion and withdrawal syringes were driven by an electric gear-driven pump (Harvard Apparatus). Artificial cerebrospinal fluid (CSF) was infused at a rate of 0.110 ml/min. Samples were collected over a 30-min period and placed in tubes containing 0.2 ml 4% cysteine, 0.03 ml 5% sodium metabisulfite and 0.1 ml of 5% EDTA. Samples were frozen and stored at-20 °C until they could be assayed. The following protocol was used for collecting samples: (1) two 30-min samples were collected and used to determine the recovery of monoamines in the assay; (2) 3 control samples, approximately 3 ml each per 30-min period were collected and used to determine basal monoamine release before drug injection; (3) either phentolamine (10/~g in 0.5/~1 saline) or saline (0.5/~1) was microinjected into the region of the NRM; and (4) 3 more 30-min samples were collected following the injection.
Histology Following these procedures, animals were decapitated and their brains removed for histological verification of brainstem injection sites. Injection sites were identified by locating the center of cannula track in the coronal section in which the track appeared in its most ventral position. The location of each injection site was plotted on camera lucida drawings of appropriate brainstem sections.
Monoamine assayprocedures The amount of NE and 5-HT released into the superfusates was determined following extraction using high-performance liquid chromatography and electrochemical detection. The first two samples were combined and divided into equal portions. Known amounts of authentic standards were added to one of these samples to determine recovery. Recovery was also determined using non-perfused CSF samples. Serotonin and NE were extracted from the superfusion samples using a modification of the method of Hammond et al. 24. The samples were applied to glass columns containing a weak cation exchange resin
248 (Bio-Rex 70,400 mesh) which had been equilibrated with 0.2 M phosphate buffer at pH 6.1. The columns were washed 3 times with 1.0 ml aliquots of 0.02 M phosphate buffer and once with 3.0 ml distilled water. Norepinephrine was eluted with 2.0 ml of 0.67 M boric acid and serotonin was eluted with 4.0 ml of 0.5 M acetic acid. The eluates containing 5-HT and NE were collected in tubes containing 25/~l of 10 mM E D T A and 5 pl of 1% cysteine and lyophilized to concentrate the amines. The samples were frozen and stored until the monoamines were quantitated using reverse-phase high-performance liquid chromatography (HPLC) and electrochemical detection according to the method of Hammond et al. 24. The HPLC mobile phase for 5-HT contained 0.07 M NaH2PO 4, 0.2 mM E D T A , and 12% methanol, and was adjusted to p H 4.8. The composition of the mobile phase for NE was similar, except for the absence of methanol and the addition of 2.0 mM heptane sulfonic acid to increase the retention time on the column. The HPLC system consisted of a solvent pump (Waters, Model 6000 A) and a reverse-phase ODS Cls column (30 cm x 3.9 mm). The flow rate was set at 1.0 ml/min. A glassy carbon thin-layer detection cell and an electrochemical detector (BioanaA
lytical Systems) were used to detect and quantitate monoamines in the effluent from the HPLC column. The detector potential was set at 0.72 V vs an AgAgCl reference electrode. The lyophilized amine samples were reconstituted in small volumes of mobile phase and injected into the HPLC system. The concentration of 5-HT and NE in the superfusates was determined using peak heights for samples and authentic standards run at the beginning and end of each assay. The values obtained were corrected for recovery which was between 70 and 90%. Some samples were pink which indicated the presence of blood. Since contamination of the samples with blood was found to increase the concentration of 5-HT, the 5-HT values for bloody samples were not included in the data analysis. Since NE concentrations are not affected by contamination with blood 24, the values for NE from these samples were included in the data analysis.
Statistical methods Statistical evaluation of the effect of microinjecting saline or phentolamine into the NRM on monoamine concentration in superfusates was done using B
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Fig. 1. Microinjection sites in the brainstem. A: sites in the NRM where phentolamine was microinjected (n = 17). B: sites outside the NRM where phentolamine was microinjected (n = 8). C: sites in the NRM were saline was microinjected (n = 8). The numbers to the right of the sections indicate the distance (mm) posterior to the interaural line.
249 one-way analysis of variance 29. Dunnett's test 29 was used to make post-hoe comparisons between the mean concentration of monoamines in the control (basal) sample taken immediately before injection into the NRM and those in the 3 samples taken after the injection. RESULTS
Effect of phentolamine injected into the NRM The mean values for basal release of 5-HT during the three 30-min samples taken before the injection of phentolamine into the NRM were 0.29 + 0.06, 0.26 + 0.09, and 0.26 + 0.08 ng/ml, respectively. Immediately following the collection of sample 3, phen-
tolamine (10/~g in 0.5 #1 saline) was microinjected into the sites illustrated in Fig. 1. These sites were primarily located within 0.5 mm of the midline and were within 0.5 mm of the dorsal surface of the pyramidal tract and corresponded to the NRM and magnocellular reticular formation dorsal to the pyramidal
tract. The injection of phentolamine into the NRM produced a marked elevation in the concentration of 5-HT in the superfusate collected within the first 30 min following the injection (Fig. 2B). The concentration of 5-HT increased from a mean value of 0.26 + 0.08 ng/ml for sample 3, collected before the injection, to 0.67 + 0.25 ng/ml in sample 4 collected after the injection. The difference between these values was statistically significant (P < 0.01, ANOVA).
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Fig. 2. Release of monoamines into spinal cord superfusates following the injection of phentolamine into the NRM. Ordinate: monoamine concentration in ng/ml. Abscissa: sample number (each sample was collected during a 30-min period of time). The arrow indicates the time at which phentolamine was injected. A: NE release following injection of phentolamine into the NRM (n = 17). B: 5-HT release following injection of phentolamine into the NRM (n = 10). Seven of the 5-HT samples contained detectable amounts of blood and were excluded from the analysis.
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Fig. 3. Release of monoamines into spinal cord superfusates following the injection of phentolamine into sites located outside the NRM. Ordinate: monoamine concentration in ng/ml. Abscissa: sample number (each sample was collected during a 30-rain period of time). The arrow indicates the time at which phentolamine was injected. A: NE release following injection of phentolamine outside the NRM (n -- 8). B: 5-HT release following injection of phentolamine outside the NRM (n = 5). Three of the 5-HT samples contained detectable amounts of blood and were excluded from the analysis.
250 However, the concentrations of 5-HT in the last two samples (5 and 6) were not significantly different from that in sample 3 (P > 0.05, ANOVA). Injection of phentolamine into the NRM also increased the concentration of NE in the superfusates collected after the injection (Fig. 2A). The concentration of NE increased from 0.29 + 0.06 ng/ml before the injection to 0.55 + 0.14 ng/ml in sample 4 collected in the 30min period after the injection. The difference between these values was statistically significant (P < 0.01, ANOVA). The concentration of NE in samples 5 and 6 was also significantly higher than that in the preinjection sample (P < 0.01, ANOVA).
Effect of phentolamine injected outside the NRM Increases in NE and 5-HT release were observed only when phentolamine was injected in the region of
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NRM or the area immediately dorsal to the pyramidal tracts. When injections were made outside this region (Fig. 1B), no statistically significant changes were observed in the concentrations of either NE (Fig. 3A) or 5-HT (Fig. 3B) in samples taken at any time following the injection of phentolamine (P > 0.05, ANOVA). These sites were located dorsolateral to the NRM in the nucleus gigantocellularis, dorsal to the NRM in the medial longitudinal fasciculus, ventrolateral to the NRM in the pyramidal tracts, and caudal to the NRM in the inferior olive.
Effect of saline injected into the NRM Saline was also microinjected into the NRM to control for possible effects due to pressure- or temperature-induced changes in neuronal excitability rather than drug-induced effects. Saline was injected in a volume of 0.05/A into the sites illustrated in Fig. 1C. These sites were similar to those into which phentolamine was injected (Fig. 1A) and were located in the NRM or in the area immediately dorsal to the pyramidal tracts. The injection of saline into these sites failed to produce any statistically significant increases (P > 0.05, A N O V A ) in the concentration of either NE or 5-HT in the samples collected after the injection. DISCUSSION
6
It is well-established that the N R M is an important
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Fig. 4. Release of monoamines into spinal cord superfusates following the injection of saline into the NRM. Ordinate: monoamine concentration in ng/ml. Abscissa: sample number. The arrow indicates the time at which saline was injected. A: NE release followinginjection of saline into the NRM (n = 8). B: 5-HT release following injection of saline into the NRM (n = 6). Two of the 5-HT samples contained detectable amounts of blood and were excluded from the analysis.
component of a brainstem monoaminergic system involved in the modulation of spinal nociceptive processes 2,3,2°,54,55. Activation of neurons in the NRM by electrical stimulation produces a decreased sensitivity to noxious stimuli 32-34'3s. Conversely, lesions of the NRM result in hyperalgesia, suggesting that neurons in this nucleus tonically inhibit the transmission of nociceptive information 36-38. The antinociception induced by activation of NRM neurons appears to be mediated by the release of both 5-HT and NE in the spinal cord. This conclusion is based on studies which have shown that activation of the NRM by electrical stimulation 1'25 or microinjection of glutamate 28 produces antinociception which can be attenuated by the intrathecal administration of monoaminergic receptor antagonists. Furthermore, electrical stimulation of the NRM, using current parameters known to produce antinociception, has been shown to increase the
251 release of endogenous NE and 5-HT into spinal cord superfusates 24. Previous reports from this and other laboratories have suggested that the activity of NRM neurons is under tonic inhibitory control by brainstem noradrenergic neurons 21'22'41'43'45'51'53. The blockade of this inhibitory NA input to NRM neurons produced by microinjecting NA antagonists results in potent antinociception, presumably due to the disinhibition (activation) of NRM neurons 41'43'45'51'53. Recent reports from this laboratory have suggested that the A 5 catecholamine nucleus may be the origin of the inhibitory NA input to the NRM, since A 5 catecholamine neurons send axonal projections to the NRM 4° and lesions in this region result in a reduction in pain sensitivity similar to that produced by NA receptor blockade in the NRM 44. The antinociception induced by blocking NA receptors in the NRM appears to be mediated by spinally projecting monoaminergic neurons, since it is reversed by the injection of either serotonergic 22 or a-adrenergic 41 antagonists into the spinal cord subarachnoid space. Furthermore, the depletion of spinal cord 5-HT and/or NE content by neurotoxins severely attenuates or blocks the induction of such antinociception45. Together, these studies support the proposal that disinhibition of NRM neurons produced by the blockade of NA receptors in this region inhibits nociceptive transmission in the spinal cord by: (1) activation of raphe spinal serotonergic neurons; and (2) activation of bulbospinal NA neurons. The purpose of the present study was to directly measure the release of endogenous NE and 5-HT in the spinal cord following the injection of the a-adrenergic antagonist phentolamine into the NRM. The major finding of these experiments was the observation that blockade of noradrenergic receptors on neurons in the NRM by the noradrenergic antagonist phentolamine produced an increase in the release of endogenous 5-HT and NE in the spinal cord. These results are similar to those reported by others using similar methods for spinal cord superfusion and quantitation of monoamines. The range of reported average values for the basal release of monoamines into spinal cord superfusates is 0.12-0.41 ng/ml for NE and 0.21-0.27 ng/ml for 5-HT 5'23'24. The values for the basal release of NE and 5-HT observed in the present experiments were within the
range of these reported values. In addition, the values for monoamine release induced by electrical stimulation of the NRM 24 are also similar to those observed in the present experiments. Thus, Hammond and coworkers24 observed increases in the release of NE and 5-HT of 214 and 157%, respectively, during electrical stimulation of NRM. Also, the addition of 80 mM potassium to the superfusion medium has been demonstrated to induce similar changes in the release of NE and 5-HT on the order of 187 and 221%, respectively57. In the present studies, activation of NRM neurons induced by microinjection of phentolamine into the NRM increased NE and 5-HT release by 189 and 257%, respectively. Thus, the results of the present studies are consistent with other reports using similar methods to demonstrate the release of monoamines into spinal cord superfusates. The active sites corresponded to the nucleus raphe magnus and the region lateral to the NRM and dorsal to the pyramidal tracts. These sites have been demonstrated to be densely innervated by catecholamine terminals and varicosities 15,21,3°,47,48. Furthermore, the areas where phentolamine was effective corresponds to the distribution of serotonin-containing perikarya demonstrated by fluorescence histochemistry12 and immunocytochemistry6-8,27,46. Some of these serotonin-containing neurons have axonal projections to the spinal cord 6-8,52 and appear to be involved in controlling nociception 2,3,2°,54,55. In addition, NA axon terminals have been demonstrated to make synaptic contacts on the dendrites and somata of serotonergic neurons located in the NRM 35. The sites at which injection of phentolamine augmented the release of 5-HT and NE in spinal cord superfusates were similar to those sites at which microinjection of phentolamine induced antinociception 21'22'41'43'45. Taken together, these data support the proposal that the antinociception induced by injection of phentolamine into the NRM is mediated by the release of 5-HT and NE in the spinal cord. Comparisons made between the time course of the elevated monoamine release and the antinociception induced by microinjection of phentolamine in the NRM, however, reveal some discrepancies between these two parameters. For example, the time course of the antinociception induced by phentolamine (10 pg in 0.5 pl of saline) is approximately 90 rain using the tail-flick test and 120 min using the hot plate 21.
252 The augmented release of N E into spinal cord superfusates produced by the injection of phentolamine in the N R M persisted for more than 90 min which correlates with the duration of the antinociception induced by phentolamine. However, the 5-HT concentration in spinal cord superfusates was significantly elevated only during the first 30 min after the phentolamine injection. These data suggest that the release of NE produced by phentolamine may be more important in producing antinociception than the release of 5-HT. It is not clear why the time course of the release of NE and 5-HT was different, but several possible explanations may be offered. For example, the release of 5-HT from presynaptic terminals in many areas of the brain has been demonstrated to be controlled by feedback inhibition mediated by presynaptic 5-HT receptors 4'9'10'13"16'19'26'50.Thus, the short duration of augmented 5-HT release following the activation of raphe spinal serotonergic neurons by phentolamine may be due to feedback inhibition of 5-HT release. Furthermore, there is evidence that 5-HT release may be inhibited by N E acting on a2-noradrenergic receptors located on the terminals of serotonergic
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neurons 14A7As'31. These data suggest that enhanced release of NE in the spinal cord produced by the injection of phentolamine in the N R M may have decreased the release of 5-HT and could explain the short duration of the augmented 5-HT release observed in these experiments. We have recently reported evidence which indicates that a similar feedback inhibitory mechanism does not control the release of N E in the spinal cord 4e. The present studies have demonstrated that chemically induced activation of N R M neurons can induce the release of endogenous 5-HT and NE into spinal cord superfusates. These observations provide direct evidence which suggests that the antinociception induced by chemical activation of neurons in the N R M is mediated by activating spinally projecting 5H T and N A neurons.
ACKNOWLEDGEMENT This work was supported by U S P H S Grant D A 03980.
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253
Physiol. Scand., Suppl., 247 (1965) 37-84. 16 Galzin, A.M., Moret, C., Verzier, B. and Langer, S.Z., Interaction between tricyclic and nontricyclic 5-hydroxytryptamine uptake inhibitors and the presynaptic 5-hydroxytryptamine inhibitory autoreceptors in the rat hypothalamus, J. Pharmacol. Exp. Ther., 235 (1985) 200-211. 17 Gothert, M. and Huth, H., Alpha-adrenoceptor-mediated modulation of 5-hydroxytryptamine release from rat brain cortex slices, Naunyn-Schmiedeberg's Arch. Pharmacol., 313 (1980) 21-26. 18 Gothert, M., Huth, H. and Schlicker, E., Characterization of the receptor subtype involved in alpha-adrenoceptormediated modulation of serotonin release from rat brain cortex slices, Naunyn-Schmiedeberg's Arch. Pharmacol., 317 (1981) 199-203. 19 Gothert, M. and Weinheimer, G., Extracellular 5-hydroxytryptamine inhibits 5-hydroxytryptamine release from rat brain cortex slices, Naunyn-Schmiedeberg's Arch. Pharmacol., 310 (1979) 93-96. 20 Hammond, D.L., Pharmacology of central pain-modulating networks (biogenic amines and nonopioid analgesics), Adv. Pain Res. Ther., 9 (1985) 499-511. 21 Hammond, D.L., Levy, R.A. and Proudfit, H.K., Hypoalgesia following microinjection of noradrenergic antagonists in the nucleus raphe magnus, Pain, 9 (1980) 85-101. 22 Hammond, D.L., Levy, R.A. and Proudfit, H.K., Hypoalgesia induced by microinjection of a norepinephrine antagonist in the raphe magnus: reversal by intrathecal administration of a serotonin antagonist, Brain Research, 201 (1980) 475-479. 23 Hammond, D.L., Tyce, G.M. and Yaksh, T.L., Drug-induced alterations in the efflux of 5-hydroxytryptamine and of 5-hydroxyindoleacetic acid into superfusates of the rat spinal cord, Eur. J. Pharmacol., 87 (1983) 441-448. 24 Hammond, D.L., Tyce, G.M. and Yaksh, T.L., Efflux of 5hydroxytryptamine and noradrenaline into spinal cord superfusates during stimulation of the rat medulla, J. Physiol. (London), 359 (1985) 151-162. 25 Hammond, D.L. and Yaksh, T.L., Antagonism of stimulation-produced antinociception by intrathecal administration of methysergide or phentolamine, Brain Research, 298 (1984) 329-337. 26 Hamon, M., Bourgoin, S., Jagger, J. and Glowinski, J., Effects of LSD on synthesis and release of 5-HT in rat brain slices, Brain Research, 69 (1974) 265-280. 27 Hrkfelt, T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M., Immunocytochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system, Neuroscience, 3 (1978) 517-538. 28 Jensen, T.S. and Yaksh, T.L., Spinal monoamine and opiate systems partly mediate the antinociceptive effects produced by glutamate at brainstem sites, Brain Research, 321 (1984) 287-297. 29 Keppel, G., Design and Analysis: A Researcher's Handbook, Prentice-Hall, Englewood Cliffs, NJ, 1973, 658 pp. 30 Levitt, P. and Moore, R.Y., Origin and organization of brainstem catecholamine innervation in the rat, J. Comp. Neurol., 186 (1979) 505-528. 31 Maura, G., Gemignani, A. and Raiteri, M., Noradrenaline inhibits central serotonin release through alpha2-adrenoceptors located on serotonergic nerve terminals, NaunynSchmiedeberg's Arch. Pharmacol., 320 (1982) 272-274.
32 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. 33 Oliveras, J.L., Hosobuchi, Y., Redjemi, F., Guilbaud, G. and Besson, J.M., Opiate antagonist, naloxone, strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior), Brain Research, 120 (1977) 221-229. 34 Oliveras, J.L., Guilbaud, G. and Besson, J.M., A map of serotonergic structures involved in stimulation producing analgesia in unrestrained freely moving cats, Brain Research, 164 (1979) 317-322. 35 Pretel, S. and Ruda, M.A., Immunocytochemical investigation of monoaminergic interaction in brain stem raphe nuclei of the cat, Soc. Neurosci. Abstr., 11 (1985) 579. 36 Proudfit, H.K., Reversible inactivation of raphe magnus neurons: effect on nociceptive threshold and morphine-induced analgesia, Brain Research, 201 (1980) 459-464. 37 Proudfit, H.K., Time-course of alterations in morphine-induced analgesia and nociceptive threshold following medullary raphe lesions, Neuroscience, 6 (1981) 945-951. 38 Proudfit, H.K. and Anderson, E.G., Morphine analgesia: blockade by raphe magnus lesions, Brain Research, 98 (1975) 612-618. 39 Proudfit, H.K. and Sagen, J., Release of endogenous norepinephrine and serotonin into spinal superfusates following local injection of phentolamine into the nucleus raphe magnus, Soc. Neurosci. Abstr., 9 (1983) 791. 40 Sagen, J. and Proudfit, H.K., Identification of noradrenergic neurons projecting to medullary nuclei involved in the modulation of pain, Soc. Neurosci. Abstr., 7 (1981) 880. 41 Sagen, J. and Proudfit, H.K., Hypoalgesia induced by blockade of noradrenergic projections to the raphe magnus: reversal by blockade of noradrenergic projections to the spinal cord, Brain Research, 223 (1981) 391-396. 42 Sagen, J. and Proudfit, H.K., Effect of intrathecally administered noradrenergic antagonists on nociception in the rat, Brain Research, 310 (1984) 295-301. 43 Sagen, J. and Proudfit, H.K., Evidence for pain modulation by pre- and postsynaptic noradrenergic receptors in the medulla oblongata, Brain Research, 331 (1985) 285-293. 44 Sagen, J. and Proudfit, H.K., Alterations in nociception following lesions of the A5 catecholamine nucleus, Brain Research, 370 (1986) 93-101. 45 Sagen, J., Winker, M.A. and Proudfit, H.K., Hypoalgesia induced by the local injection of phentolamine in the nucleus raphe magnus: blockade by depletion of spinal cord monoamines, Pain, 16 (1983) 253-263. 46 Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat - - cell bodies and terminals, Neuroscience, 6 (1981) 557-618. 47 Swanson, L.W. and Hartman, B.K., The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-fl-hydroxylase as a marker, J. Comp. Neurol., 163 (1975) 467-506. 48 Takagi, H., Yamamoto, K., Shiosaka, S., Senba, E., Takatsuki, K., Inagaki, S., Sakanaka, M. and Yohyama, M., Morphological study of noradrenaline innervation in the caudal raphe nuclei with special reference to fine structure, J. Comp. Neurol., 203 (1981) 15-22. 49 Tyce, G.M. and Yaksh, T.L., Monoamine release from cat spinal cord by somatic stimuli: an intrinsic modulatory system, J. Physiol. (London), 314 (1981) 513-529.
254 50 Verbeuren, T.J., Coen, E.P., Schoups, A., Van de Velde, R., Baeyens, R. and De Potter, W.P., Presynaptic serotonin receptors regulate the release of 3H-serotonin in hypothalamic slices of the rabbit, Naunyn-Schmiedeberg's Arch. Pharmacol., 327 (1984) 102-106. 51 Wessendorf, M.W. and Anderson, E.G., Single unit studies of identified bulbospinal serotonergic units, Brain Research, 279 (1983) 93-103. 52 Wessendorf, M.W., Proudfit, H.K. and Anderson, E.G., The identification of serotonergic neurons in the nucleus raphe magnus by conduction velocity, Brain Research, 214 (1981) 168-173. 53 Willcockson, K.D., Gerhart, C.L., Cargill, C.L. and Willis, W.D., Effects of biogenic amines on raphe-spinal tract cells, J. Pharmacol. Exp. Ther., 225 (1983) 637-645.
54 Willis, W.D., Control of nociceptive transmission in the spinal cord. In H. Autrum, D.O. Ottoson, E.R. Perl and R.F. Schmidt (Eds.), Progress in Sensory Physiology, Springer, New York, 1982. 55 Yaksh, T.L. and Hammond, D.L., Putative transmitters of descending systems - - studies of local administration and liberation. In Brain Stem Control of Spinal Mechanisms, Elsevier, Amsterdam, 1982, pp. 473-492. 56 Yaksh, T.L. and Tyce, G.M., Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from spinal cord, Brain Research, 171 (1979) 176-181. 57 Yaksh, T.L. and Tyce, G.M., Resting and K÷-evoked release of serotonin and norepinephrine in vivo from the rat and cat spinal cord, Brain Research, 192 (1980) 133-146.
Braip~Rese~lrch, 4t)6 (1987) -~4 6 - _~'~q4 Elscvicr
BRE 12437
Release of endogenous monoamines into spinal cord superfusates following the microinjection of phentolamine into the nucleus raphe magnus Jacqueline Sagen and Herbert K. Proudfit Department of Pharmacology, University of lllinois College of Medicine at Chicago, Chicago, 1L 60680 ( U.S.A ) (Accepted 12 August 1986) Key words: Antinociception; Nucleus raphe magnus; Norepinephrine; Serotonin; Spinal cord; Superfusion; Neurotransmitter release
Previous studies have suggested that raphe-spinal neurons located in the nucleus raphe magnus (NRM) are tonically inhibited by noradrenergic neurons. Furthermore, blockade of the inhibitory noradrenergic input to the NRM induces antinociception which appears to be mediated by the release of both serotonin and norepinephrine in the spinal cord. The present experiments were designed to directly measure the release of endogenous serotonin and norepinephrine into spinal cord superfusates before and after the microinjection of the a-adrenergic antagonist phentolamine into the NRM. High-performance liquid chromatography with electrochemical detection was used to quantitate the monoamines. The injection of phentolamine into the NRM induced a significant increase in the amount of both norepinephrine and serotonin released in the spinal cord. This enhanced release was not observed following either the injection of phentolamine into sites outside the NRM or the injection of saline vehicle into the NRM. These results support the proposal that the antinociception induced by the blockade of the inhibitory noradrenergic input to the NRM is mediated by the activation of spinally-projecting serotonergic and noradrenergic neurons.
INTRODUCTION It is well established that neurons in the nucleus raphe magnus (NRM) play an important role in modulating the perception of noxious stimuli 3'2°'54'55. The activity of these N R M neurons appears to be under the tonic inhibitory control of brainstem N A neurons 51,53. Blockade of this inhibitory N A input by the microinjection of a-adrenergic antagonists induces potent antinociception 21'22'41'43'45. Previous studies have suggested that this antinociception is mediated by spinally-projecting monoaminergic neurons, since it is reversed by the injection of either serotonergic 22 or a-adrenergic 41 antagonists into the spinal cord subarachnoid space. Furthermore, the depletion of spinal cord serotonin (5-HT) and/or norepinephrine (NE) content by neurotoxins severely attenuates or blocks the induction of such antinociception 45.
Together, these studies support the proposal that disinhibition of N R M neurons produced by the blockade of N A receptors in this region inhibits nociceptive transmission in the spinal cord by: (1) activation of raphe-spinal serotenergic neurons; and (2) activation of bulbospinal N A neurons. This implies that such antinociception results from the release of both 5-HT and N E from axon terminals in the spinal cord. Recent studies have demonstrated that it is possible to measure changes in the concentration of these monoamines in spinal cord superfusates following pharmacological and physiological manipulations 24,49,56,57. The purpose of this study was to directly measure the release of endogenous N E and 5-HT in the spinal cord following the injection of the a-adrenergic antagonist phentolamine into the NRM. A preliminary account of these experiments has been reported 39.
Correspondence: H.K. Proudfit, Department of Pharmacology, University of IUinois College of Medicine at Chicago, P.O. Box 6998, Chicago, IL 60680, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
247 MATERIALSAND METHODS
Surgicalprocedures Sprague-Dawley derived rats weighing 350-400 g were surgically prepared for drug microinjection and spinal cord superfusion. A chronic indwelling stainless steel microinjection guide tube (23-gauge) aimed at the NRM (P 2.5 mm; H - 0 . 5 mm; L 0.00 mm; incisor bar -2.5 mm) was implanted at least one week prior to superfusion procedures (for details see Sagen and Proudfit43). Animals that exhibited any obvious behavioral or motor abnormalities following cannula insertion were not used. The remaining animals were housed individually and allowed free access to food and water.
Microin]ectionprocedures Drug solutions were microinjected through a 30gauge stainless steel injection cannula inserted into the indwelling guide tube. The tip of the injection cannula extended 4 mm beyond the end of the guide tube to minimize damage to the injection site. The injection cannula was connected to an electrical geardriven syringe pump (Harvard Apparatus) by a length of tubing (PE 20) filled with drug solution. The volume of each injection was 0.5/~1 delivered over a period of 60 s and monitored by observing the movement of an air bubble over a calibrated distance in the tubing. The injection cannula was left in place for 60 s after the injection to minimize flow of the drug solution up the cannula track.
Spinal cord superfusion procedures Spinal cord superfusion procedures were performed 1 week following cannula insertion. Animals were anesthetized with urethane (1200 mg/kg), fitted with a tracheal tube and immobilized in a stereotaxic head holder. An intrathecal catheter made of PE 10 tubing was threaded down the intrathecal space through a small slit in the atlanto-occipital membrane to the rostral tip of the lumbar enlargement. The catheter was connected to a 20-ml glass syringe containing artificial cerebrospinal fluid and served to infuse fluid. The outflow from the spinal cord was collected using a length of polyethylene tubing (PE 50) which was flared at the end and placed over the opening in the dura. This tubing was connected to a 3-way stopcock so that the outflow could be directed to one
of two collection tubes (50 mm long glass tubing, 3 mm internal diameter) which were kept cold on an ice slurry. These tubes were connected to a second 20-ml glass syringe. This arrangement allowed sequential samples to be collected without stopping the flow of the superfusion. The infusion and withdrawal syringes were driven by an electric gear-driven pump (Harvard Apparatus). Artificial cerebrospinal fluid (CSF) was infused at a rate of 0.110 ml/min. Samples were collected over a 30-min period and placed in tubes containing 0.2 ml 4% cysteine, 0.03 ml 5% sodium metabisulfite and 0.1 ml of 5% EDTA. Samples were frozen and stored at-20 °C until they could be assayed. The following protocol was used for collecting samples: (1) two 30-min samples were collected and used to determine the recovery of monoamines in the assay; (2) 3 control samples, approximately 3 ml each per 30-min period were collected and used to determine basal monoamine release before drug injection; (3) either phentolamine (10/~g in 0.5/~1 saline) or saline (0.5/~1) was microinjected into the region of the NRM; and (4) 3 more 30-min samples were collected following the injection.
Histology Following these procedures, animals were decapitated and their brains removed for histological verification of brainstem injection sites. Injection sites were identified by locating the center of cannula track in the coronal section in which the track appeared in its most ventral position. The location of each injection site was plotted on camera lucida drawings of appropriate brainstem sections.
Monoamine assayprocedures The amount of NE and 5-HT released into the superfusates was determined following extraction using high-performance liquid chromatography and electrochemical detection. The first two samples were combined and divided into equal portions. Known amounts of authentic standards were added to one of these samples to determine recovery. Recovery was also determined using non-perfused CSF samples. Serotonin and NE were extracted from the superfusion samples using a modification of the method of Hammond et al. 24. The samples were applied to glass columns containing a weak cation exchange resin
248 (Bio-Rex 70,400 mesh) which had been equilibrated with 0.2 M phosphate buffer at pH 6.1. The columns were washed 3 times with 1.0 ml aliquots of 0.02 M phosphate buffer and once with 3.0 ml distilled water. Norepinephrine was eluted with 2.0 ml of 0.67 M boric acid and serotonin was eluted with 4.0 ml of 0.5 M acetic acid. The eluates containing 5-HT and NE were collected in tubes containing 25/~l of 10 mM E D T A and 5 pl of 1% cysteine and lyophilized to concentrate the amines. The samples were frozen and stored until the monoamines were quantitated using reverse-phase high-performance liquid chromatography (HPLC) and electrochemical detection according to the method of Hammond et al. 24. The HPLC mobile phase for 5-HT contained 0.07 M NaH2PO 4, 0.2 mM E D T A , and 12% methanol, and was adjusted to p H 4.8. The composition of the mobile phase for NE was similar, except for the absence of methanol and the addition of 2.0 mM heptane sulfonic acid to increase the retention time on the column. The HPLC system consisted of a solvent pump (Waters, Model 6000 A) and a reverse-phase ODS Cls column (30 cm x 3.9 mm). The flow rate was set at 1.0 ml/min. A glassy carbon thin-layer detection cell and an electrochemical detector (BioanaA
lytical Systems) were used to detect and quantitate monoamines in the effluent from the HPLC column. The detector potential was set at 0.72 V vs an AgAgCl reference electrode. The lyophilized amine samples were reconstituted in small volumes of mobile phase and injected into the HPLC system. The concentration of 5-HT and NE in the superfusates was determined using peak heights for samples and authentic standards run at the beginning and end of each assay. The values obtained were corrected for recovery which was between 70 and 90%. Some samples were pink which indicated the presence of blood. Since contamination of the samples with blood was found to increase the concentration of 5-HT, the 5-HT values for bloody samples were not included in the data analysis. Since NE concentrations are not affected by contamination with blood 24, the values for NE from these samples were included in the data analysis.
Statistical methods Statistical evaluation of the effect of microinjecting saline or phentolamine into the NRM on monoamine concentration in superfusates was done using B
C
4.0
4,5
5.0
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Fig. 1. Microinjection sites in the brainstem. A: sites in the NRM where phentolamine was microinjected (n = 17). B: sites outside the NRM where phentolamine was microinjected (n = 8). C: sites in the NRM were saline was microinjected (n = 8). The numbers to the right of the sections indicate the distance (mm) posterior to the interaural line.
249 one-way analysis of variance 29. Dunnett's test 29 was used to make post-hoe comparisons between the mean concentration of monoamines in the control (basal) sample taken immediately before injection into the NRM and those in the 3 samples taken after the injection. RESULTS
Effect of phentolamine injected into the NRM The mean values for basal release of 5-HT during the three 30-min samples taken before the injection of phentolamine into the NRM were 0.29 + 0.06, 0.26 + 0.09, and 0.26 + 0.08 ng/ml, respectively. Immediately following the collection of sample 3, phen-
tolamine (10/~g in 0.5 #1 saline) was microinjected into the sites illustrated in Fig. 1. These sites were primarily located within 0.5 mm of the midline and were within 0.5 mm of the dorsal surface of the pyramidal tract and corresponded to the NRM and magnocellular reticular formation dorsal to the pyramidal
tract. The injection of phentolamine into the NRM produced a marked elevation in the concentration of 5-HT in the superfusate collected within the first 30 min following the injection (Fig. 2B). The concentration of 5-HT increased from a mean value of 0.26 + 0.08 ng/ml for sample 3, collected before the injection, to 0.67 + 0.25 ng/ml in sample 4 collected after the injection. The difference between these values was statistically significant (P < 0.01, ANOVA).
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Fig. 2. Release of monoamines into spinal cord superfusates following the injection of phentolamine into the NRM. Ordinate: monoamine concentration in ng/ml. Abscissa: sample number (each sample was collected during a 30-min period of time). The arrow indicates the time at which phentolamine was injected. A: NE release following injection of phentolamine into the NRM (n = 17). B: 5-HT release following injection of phentolamine into the NRM (n = 10). Seven of the 5-HT samples contained detectable amounts of blood and were excluded from the analysis.
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Fig. 3. Release of monoamines into spinal cord superfusates following the injection of phentolamine into sites located outside the NRM. Ordinate: monoamine concentration in ng/ml. Abscissa: sample number (each sample was collected during a 30-rain period of time). The arrow indicates the time at which phentolamine was injected. A: NE release following injection of phentolamine outside the NRM (n -- 8). B: 5-HT release following injection of phentolamine outside the NRM (n = 5). Three of the 5-HT samples contained detectable amounts of blood and were excluded from the analysis.
250 However, the concentrations of 5-HT in the last two samples (5 and 6) were not significantly different from that in sample 3 (P > 0.05, ANOVA). Injection of phentolamine into the NRM also increased the concentration of NE in the superfusates collected after the injection (Fig. 2A). The concentration of NE increased from 0.29 + 0.06 ng/ml before the injection to 0.55 + 0.14 ng/ml in sample 4 collected in the 30min period after the injection. The difference between these values was statistically significant (P < 0.01, ANOVA). The concentration of NE in samples 5 and 6 was also significantly higher than that in the preinjection sample (P < 0.01, ANOVA).
Effect of phentolamine injected outside the NRM Increases in NE and 5-HT release were observed only when phentolamine was injected in the region of
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NRM or the area immediately dorsal to the pyramidal tracts. When injections were made outside this region (Fig. 1B), no statistically significant changes were observed in the concentrations of either NE (Fig. 3A) or 5-HT (Fig. 3B) in samples taken at any time following the injection of phentolamine (P > 0.05, ANOVA). These sites were located dorsolateral to the NRM in the nucleus gigantocellularis, dorsal to the NRM in the medial longitudinal fasciculus, ventrolateral to the NRM in the pyramidal tracts, and caudal to the NRM in the inferior olive.
Effect of saline injected into the NRM Saline was also microinjected into the NRM to control for possible effects due to pressure- or temperature-induced changes in neuronal excitability rather than drug-induced effects. Saline was injected in a volume of 0.05/A into the sites illustrated in Fig. 1C. These sites were similar to those into which phentolamine was injected (Fig. 1A) and were located in the NRM or in the area immediately dorsal to the pyramidal tracts. The injection of saline into these sites failed to produce any statistically significant increases (P > 0.05, A N O V A ) in the concentration of either NE or 5-HT in the samples collected after the injection. DISCUSSION
6
It is well-established that the N R M is an important
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Fig. 4. Release of monoamines into spinal cord superfusates following the injection of saline into the NRM. Ordinate: monoamine concentration in ng/ml. Abscissa: sample number. The arrow indicates the time at which saline was injected. A: NE release followinginjection of saline into the NRM (n = 8). B: 5-HT release following injection of saline into the NRM (n = 6). Two of the 5-HT samples contained detectable amounts of blood and were excluded from the analysis.
component of a brainstem monoaminergic system involved in the modulation of spinal nociceptive processes 2,3,2°,54,55. Activation of neurons in the NRM by electrical stimulation produces a decreased sensitivity to noxious stimuli 32-34'3s. Conversely, lesions of the NRM result in hyperalgesia, suggesting that neurons in this nucleus tonically inhibit the transmission of nociceptive information 36-38. The antinociception induced by activation of NRM neurons appears to be mediated by the release of both 5-HT and NE in the spinal cord. This conclusion is based on studies which have shown that activation of the NRM by electrical stimulation 1'25 or microinjection of glutamate 28 produces antinociception which can be attenuated by the intrathecal administration of monoaminergic receptor antagonists. Furthermore, electrical stimulation of the NRM, using current parameters known to produce antinociception, has been shown to increase the
251 release of endogenous NE and 5-HT into spinal cord superfusates 24. Previous reports from this and other laboratories have suggested that the activity of NRM neurons is under tonic inhibitory control by brainstem noradrenergic neurons 21'22'41'43'45'51'53. The blockade of this inhibitory NA input to NRM neurons produced by microinjecting NA antagonists results in potent antinociception, presumably due to the disinhibition (activation) of NRM neurons 41'43'45'51'53. Recent reports from this laboratory have suggested that the A 5 catecholamine nucleus may be the origin of the inhibitory NA input to the NRM, since A 5 catecholamine neurons send axonal projections to the NRM 4° and lesions in this region result in a reduction in pain sensitivity similar to that produced by NA receptor blockade in the NRM 44. The antinociception induced by blocking NA receptors in the NRM appears to be mediated by spinally projecting monoaminergic neurons, since it is reversed by the injection of either serotonergic 22 or a-adrenergic 41 antagonists into the spinal cord subarachnoid space. Furthermore, the depletion of spinal cord 5-HT and/or NE content by neurotoxins severely attenuates or blocks the induction of such antinociception45. Together, these studies support the proposal that disinhibition of NRM neurons produced by the blockade of NA receptors in this region inhibits nociceptive transmission in the spinal cord by: (1) activation of raphe spinal serotonergic neurons; and (2) activation of bulbospinal NA neurons. The purpose of the present study was to directly measure the release of endogenous NE and 5-HT in the spinal cord following the injection of the a-adrenergic antagonist phentolamine into the NRM. The major finding of these experiments was the observation that blockade of noradrenergic receptors on neurons in the NRM by the noradrenergic antagonist phentolamine produced an increase in the release of endogenous 5-HT and NE in the spinal cord. These results are similar to those reported by others using similar methods for spinal cord superfusion and quantitation of monoamines. The range of reported average values for the basal release of monoamines into spinal cord superfusates is 0.12-0.41 ng/ml for NE and 0.21-0.27 ng/ml for 5-HT 5'23'24. The values for the basal release of NE and 5-HT observed in the present experiments were within the
range of these reported values. In addition, the values for monoamine release induced by electrical stimulation of the NRM 24 are also similar to those observed in the present experiments. Thus, Hammond and coworkers24 observed increases in the release of NE and 5-HT of 214 and 157%, respectively, during electrical stimulation of NRM. Also, the addition of 80 mM potassium to the superfusion medium has been demonstrated to induce similar changes in the release of NE and 5-HT on the order of 187 and 221%, respectively57. In the present studies, activation of NRM neurons induced by microinjection of phentolamine into the NRM increased NE and 5-HT release by 189 and 257%, respectively. Thus, the results of the present studies are consistent with other reports using similar methods to demonstrate the release of monoamines into spinal cord superfusates. The active sites corresponded to the nucleus raphe magnus and the region lateral to the NRM and dorsal to the pyramidal tracts. These sites have been demonstrated to be densely innervated by catecholamine terminals and varicosities 15,21,3°,47,48. Furthermore, the areas where phentolamine was effective corresponds to the distribution of serotonin-containing perikarya demonstrated by fluorescence histochemistry12 and immunocytochemistry6-8,27,46. Some of these serotonin-containing neurons have axonal projections to the spinal cord 6-8,52 and appear to be involved in controlling nociception 2,3,2°,54,55. In addition, NA axon terminals have been demonstrated to make synaptic contacts on the dendrites and somata of serotonergic neurons located in the NRM 35. The sites at which injection of phentolamine augmented the release of 5-HT and NE in spinal cord superfusates were similar to those sites at which microinjection of phentolamine induced antinociception 21'22'41'43'45. Taken together, these data support the proposal that the antinociception induced by injection of phentolamine into the NRM is mediated by the release of 5-HT and NE in the spinal cord. Comparisons made between the time course of the elevated monoamine release and the antinociception induced by microinjection of phentolamine in the NRM, however, reveal some discrepancies between these two parameters. For example, the time course of the antinociception induced by phentolamine (10 pg in 0.5 pl of saline) is approximately 90 rain using the tail-flick test and 120 min using the hot plate 21.
252 The augmented release of N E into spinal cord superfusates produced by the injection of phentolamine in the N R M persisted for more than 90 min which correlates with the duration of the antinociception induced by phentolamine. However, the 5-HT concentration in spinal cord superfusates was significantly elevated only during the first 30 min after the phentolamine injection. These data suggest that the release of NE produced by phentolamine may be more important in producing antinociception than the release of 5-HT. It is not clear why the time course of the release of NE and 5-HT was different, but several possible explanations may be offered. For example, the release of 5-HT from presynaptic terminals in many areas of the brain has been demonstrated to be controlled by feedback inhibition mediated by presynaptic 5-HT receptors 4'9'10'13"16'19'26'50.Thus, the short duration of augmented 5-HT release following the activation of raphe spinal serotonergic neurons by phentolamine may be due to feedback inhibition of 5-HT release. Furthermore, there is evidence that 5-HT release may be inhibited by N E acting on a2-noradrenergic receptors located on the terminals of serotonergic
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ACKNOWLEDGEMENT This work was supported by U S P H S Grant D A 03980.
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