Skip Navigation LinksHome > May 2002 - Volume 96 - Issue 5 > Intracerebroventricular Morphine Produces Antinociception by...
Anesthesiology:
Laboratory Investigations

Intracerebroventricular Morphine Produces Antinociception by Evoking γ-Aminobutyric Acid Release through Activation of 5-Hydroxytryptamine 3 Receptors in the Spinal Cord

Kawamata, Tomoyuki M.D.*; Omote, Keiichi M.D.†; Toriyabe, Masaki M.D.‡; Kawamata, Mikito M.D.§; Namiki, Akiyoshi M.D.∥

Free Access
Article Outline
Collapse Box

Author Information

Collapse Box

Abstract

Background: It has been generally considered that supraspinal morphine activates the serotonergic descending inhibitory system and releases serotonin (5-hydroxytryptamine [5-HT]) in the spinal cord, producing antinociception through activation of 5-HT receptors. The involvement of a spinal γ-aminobutyric acid–mediated (GABAergic) system is also suggested in supraspinal morphine antinociception. It has been reported that spinal GABAergic system contributes to 5-HT3 receptor-mediated antinociception. In this study, the authors investigated the contribution of spinal 5-HT3 receptor and the GABAergic system in the intracerebroventricular morphine–induced antinociception.
Methods: Male Sprague-Dawley rats were used. Using the spinal microdialysis method, concentrations of 5-HT and GABA were measured after intracerebroventricular morphine administration. The effect of intracerebroventricular naloxone or spinal perfusion of a selective 5-HT3 receptor antagonist 3-tropanyl-indole-3-carboxylate methiodide on the spinal release of GABA after intracerebroventricular morphine administration was also examined. In the behavioral study, involvement of 5-HT3 receptors or GABAA receptors in the intracerebroventricular morphine–induced antinociceptive effect was investigated using the tail-flick test.
Results: Intracerebroventricular morphine (40 nmol) significantly increased spinal GABA and 5-HT release. Evoked spinal GABA release was reversed by intracerebroventricular naloxone (40 nmol) or spinal perfusion of 3-tropanyl-indole-3-carboxylate methiodide (1 mm). In the behavioral study, intracerebroventricular morphine produced significant antinociception. Intrathecal administration of either GABAA receptor antagonist bicuculine or 3-tropanyl-indole-3-carboxylate methiodide but not vehicle reversed the morphine-induced antinociceptive effect.
Conclusion: Intracerebroventricular morphine evokes spinal GABA release via the activation of 5-HT3 receptors in the spinal cord, resulting in antinociceptive effect.
IT is generally considered that supraspinal morphine produces its antinociceptive effect through the descending serotonergic and noradrenergic neurons projecting to spinal cord. Several spinal mechanisms of supraspinal morphine–induced antinociception have been proposed. In addition to direct inhibition of spinal neurons or primary afferent nerve terminals by noradrenaline and serotonin (5-hydroxytryptamine [5-HT]), indirect mechanisms via spinal interneurons have been suggested. 1,2 It has been reported that a spinal γ-aminobutyric acid type A (GABAA) receptor antagonist inhibits the supraspinal morphine–induced antinociception. 1 Furthermore, a spinal GABA-mediated (GABAergic) system contributes to spinally administered 5-HT3 receptor agonist–mediated antinociception. 3 These observations suggest a possible interaction between the serotonergic descending system and a spinal GABA system in supraspinal morphine–induced antinociception.
Among 5-HT receptor subtypes, only the 5-HT3 receptor is a ligand-gated cation channel. 4 Electrical stimulation in the periaqueductal gray (PAG) produces inhibition of dorsal horn neuronal responses, and this PAG-induced inhibition is blocked by spinally administered 5-HT3 receptor antagonists. 5 Behavioral studies indicated that intrathecal 5-HT–induced antinociception was mediated by 5-HT3 receptors. 6 However, it is unlikely that the activation of 5-HT3 receptors directly inhibits neuronal responses because the activation of 5-HT3 receptors causes a depolarization by means of increased membrane permeability to monovalent cation. 4 Supraspinal morphine could evoke GABA release from interneurons through the activation of 5-HT3 receptors in the spinal cord, resulting in antinociception. However, there has been no direct evidence of supraspinal morphine–induced 5-HT3 receptor activation and GABA release in the spinal cord.
In the current study, we examined whether supraspinally administered morphine would evoke GABA release through the activation of 5-HT3 receptors in the spinal cord. We also examined the functional roles of these neurotransmitters in supraspinal morphine–induced antinociception.
Back to Top | Article Outline

Materials and Methods

The protocol for this study was approved by Sapporo Medical University Animal Care and Use Committee (Sapporo, Japan). Experiments were conducted on male Sprague-Dawley rats (weight, 250–350 g; Japan SLC, Hamamatsu, Japan) housed individually in a temperature-controlled (21 ± 1°C) room with a 12-h light–dark cycle and given free access to food and water. Each animal was used in only one experiment.
Back to Top | Article Outline
Intracerebroventricular Cannula and Lumbar Intrathecal Catheter
Rats were anesthetized with pentobarbital (50 mg/kg administered intraperitoneally) and were placed in a stereotaxic head holder to implant an intracerebroventricular guide cannula. The skull was exposed, and a guide cannula equipped with a 32-guage stylet was directed into the left lateral ventricle according to Paxinos and Watson 7 (anterior-posterior +0.8 mm, midline +1.4 mm, dorsal-ventral 3.8 mm with respect to bregma) and was attached to the skull with three anchoring screws and cemented in place. For intracerebroventricular injection, the stylet was removed and the injection cannula was inserted.
For the behavioral study, a lumbar intrathecal catheter was implanted in the rats with an intracerebroventricular guide cannula. A polyethylene intrathecal catheter (PE-10; Becton Dickenson, Franklin Lakes, NJ) was inserted 15 mm cephalad into the lumbar subarachnoid space at the L4–L5 intervertebral level, with the tip of the catheter located near the lumbar enlargement of the spinal cord to administer the drugs intrathecally. The catheter was tunneled subcutaneously and externalized through the skin in the neck region. At least 6 days of postsurgical recovery were allowed before animals were used in the behavioral study.
Back to Top | Article Outline
Microdialysis Catheter Implantation
The spinal cord dialysis probe was prepared according to our modification of the method described by Skilling et al.8 The dialysis probe was constructed from a 1-cm length of dialysis fiber (an ID of 200 μm, an OD of 220 μm, and 50-kd molecular weight cutoff; DM-22, Eicom Co., Kyoto, Japan) which was coated with a thin layer of epoxy glue (Devcon Co., Danvers, MA) along the whole length, except for a 2-mm region in the middle. To make the fiber firm enough for implantation, a Nichrome-Formvar wire with a 78-μm ID (A-M Systems, Inc., Everret, WA) was passed through the fiber. Each end of the fiber was attached to 2-cm polyethylene catheters (PE-10; Becton Dickinson, Franklin Lakes, NJ), and each end of the polyethylene catheters was then attached to an 8-cm teflon tube with an ID of 100 μm and an OD of 400 μm (JT-10; Eicom). Seven days after an intracerebroventricular guide cannula implantation, rats were anesthetized with pentobarbital (50 mg/kg administered intraperitoneally) and incised along the dorsal midline from T2 to L2. The lateral surfaces of vertebra L1 were exposed, and bilateral holes were carefully made through the bone, exposing the spinal cord laterally at the level of the dorsal horn. A dialysis tube was placed through the holes by hand, passing transversely through the dorsal spinal cord. The two distal ends of the probe were tunneled subcutaneously and externalized through the skin in the neck region. The experiments were performed 18–24 h after the implantation of the dialysis probe. After a recovery period, the animals showing any signs of limb paralysis or impaired movement were excluded in this study. After each experiment, we perfused methylene blue dye through the dialysis probe to verify the position of the dialysis fiber, and then rats were killed with an overdose of pentobarbital. The data to be reported are from rats in which methylene blue dye remained in the area of the dorsal half of the dorsal horn.
Back to Top | Article Outline
Microdialysis Study
The animals freely moved in a plastic cage with dimensions of 30 × 30 × 35 cm during the dialysis experiments. A liquid switch (SI-50; Eicom) was placed between the syringe pump and the dialysis probe to enable a different drug to be administered locally via the probe. The dialysis probe was perfused with artificial cerebrospinal fluid (ACSF; 140 mm NaCl, 4.0 mm KCl, 1.26 mm CaCl2, 1.15 mm MgCl2, 2.0 mm Na2HPO4, 0.5 mm NaH2PO4, pH 7.4) at a constant flow rate of 3 μl/min. The samples were collected as 20-min fractions and divided into two samples: 20 μl dialysate collected for analysis of GABA and 20 μl for 5-HT. The samples were frozen at −80°C until analysis. Three consecutive samples were collected for determination of basal concentrations 180 min after starting perfusion of ACSF. After obtaining three consecutive samples, 40 nmol/3 μl of intracerebroventricular morphine (Sankyo Co., Tokyo, Japan) was administered over a 120-s period. The samples were collected up to 360 min after morphine administration. To examine the naloxone sensitivity and the involvement of spinal 5-HT3 receptor activation in morphine-induced spinal GABA release, either intracerebroventricular naloxone (Sigma, St.Louis, MO) or the selective 5-HT3 receptor antagonist, 3-tropanyl-indole-3-carboxylate methiodide (TICM; Research Biochemical International, Natick, MA) was administered 180 min after morphine administration. Naloxone 40 nmol was injected in a volume of 3 μl over a 120-s period. TICM (1 mm) was perfused through the dialysis catheter for 60 min. All drugs used in microdialysis study were prepared in ACSF.
Back to Top | Article Outline
Analysis of Serotonin
Serotonin in the dialysate was analyzed using high-performance liquid chromatography with electrochemical detection (Degasser, DG-100; liquid chromatograph, EP-100; electrochemical detector, ECD-100; Eicom). The chromatographic conditions were as follows: column, Eicompak (CA-5ODS 2.1 × 150 mm; Eicom); mobile phase, 0.1 m phosphate buffer (pH 6.0) containing 20.0% methanol, 0.02 mm EDTA, and 0.72 mm sodium 1-octanesulfonate for the analysis of 5-HT; working electrode, glassy carbon (WE-3G, Eicom); flow rate, 0.23 ml/min. Detector voltage and detector temperature were set at 0.45 V and at 25.0°C, respectively. Retention time for 5-HT was 14.85 min. The detection limit for 5-HT analysis is 0.05 fg/20 μl.
Back to Top | Article Outline
Analysis of γ-Aminobutyric Acid
γ-Aminobutyric acid in the dialysate was analyzed using high-performance liquid chromatography with fluorescence detection (Degasser, DG-100; liquid chromatograph, EP-100; fluorescence detector, FLD-370; Eicom) after derivatization with o-phthaldialdehyde (Sigma). The o-phthaldialdehyde derivatizing reagent was prepared by dissolving o-phthaldialdehyde (54 mg) in absolute methanol (1 ml) and adding 2-mercaptoethanol (40 μl) and sodium carbonate (0.1 m, pH 9.5, 99 ml). Ten microliters of this derivatizing reagent was mixed with 20 μl of the dialysate and allowed to react for 2.5 min. Twenty microliters of this solution was analyzed. The chromatographic conditions were as follows: column, Eicompak (SC-5ODS 2.1 × 150 mm; Eicom); mobile phase, 0.1 m sodium dihydrogenphosphate and 0.1 m disodium hydrogenphosphate (pH 3.5) containing 50.0% methanol and 0.1 mm disodium EDTA; flow rate, 0.23 ml/min. Detector temperature was set at 30°C. Retention time for GABA was 14.83 min. The detection limit for GABA analysis is 0.2 pg/20 μl.
Back to Top | Article Outline
Behavioral Study
A behavioral study was performed separately from the microdialysis study. The tail-flick test was used to assess thermal nociceptive threshold. Tail-flick testing was performed by monitoring latency to withdrawal from the heat source (a 50-W projection lamp bulb) focused on a distal segment of the tail, using a thermal analgesimeter (KN-205E; Natsume, Tokyo, Japan). A cutoff time of 15.0 s was used to minimize damage to the skin of the tail. After determination of baseline tail-flick latencies, 40 nmol/3 μl of intracerebroventricular morphine was administered. The tail-flick latencies were measured at 30, 60, 120, 180, 240, 300, and 360 min after the intracerebroventricular injection. To examine the involvement of spinal 5-HT3 receptors and GABAA receptors in morphine analgesia, either TICM or a GABAA receptor antagonist bicuculline (Sigma) was intrathecally administered 170 min after morphine administration, and the effect of the antagonist on tail-flick latencies was evaluated 10 min later. TICM and bicuculline were freshly dissolved in ACSF in concentrations that allowed intrathecal injections in 10-μl volumes. Intrathecal drug administration was accomplished by using a microinjection syringe (Hamilton, Reno, NV) connected to the intrathecal catheter in awake, briefly restrained rats. All intrathecal drugs were administered manually over 10 s.
Back to Top | Article Outline
Data Analysis
In the microdialysis study, the changes of GABA and 5-HT concentrations are presented as mean ± SD of percentage of basal concentrations. Data were compared with basal concentration using a one-way analysis of variance for repeated measures followed by Dunnett test within a single group, and were analyzed using a two-way analysis of variance for repeated measures followed by the Tukey Kramer test for between-group comparisons. In the behavioral study on morphine antinociception, the values of withdrawal latency were converted to percent maximum possible effect:
Equation U1
Equation U1
Image Tools
MATH
Changes in percent maximum possible effect were compared with baseline using a one-way analysis of variance for repeated measures followed by Dunnett test within a single group, and analyzed using a paired t test for the comparison between pretreatment and posttreatment in the behavioral study. P < 0.05 was considered statistically significant.
Back to Top | Article Outline

Results

Microdialysis Study
Serotonin Analysis.
Fig. 1
Fig. 1
Image Tools
The basal concentration of 5-HT was 2.57 ± 1.18 pg/20 μl. The coefficient of variation among the three consecutive samples for determination of basal concentrations was less than 7%. Intracerebroventricular morphine (40 nmol) evoked a significant increase in spinal 5-HT concentration (P < 0.01;fig. 1). Significant increases in 5-HT concentration were observed 40–240 min after morphine administration.
Back to Top | Article Outline
γ-Aminobutyric Acid Analysis.
Fig. 2
Fig. 2
Image Tools
Fig. 3
Fig. 3
Image Tools
The basal concentration of GABA was 0.27 ± 0.14 pg/20 μl. The coefficient of variation among the three consecutive samples for determination of basal concentrations was less than 5%. Figure 2 shows the effect of 40 nmol intracerebroventricular morphine on spinal GABA concentration. GABA concentration was gradually increased after intracerebroventricular morphine administration and reached a plateau from 180 to 260 min after morphine administration. Significant increases in GABA concentration were observed from 80 up to 360 min after administration of morphine compared with the basal concentration (P < 0.01). Intracerebroventricular naloxone (40 nmol) administered at 180 min after morphine significantly reversed the morphine-induced increase in GABA concentration (P < 0.01). Spinal perfusion of 1 mm TICM for 60 min (from 180 to 240 min after morphine) also reversed morphine-induced increase in GABA concentration (P < 0.01). GABA concentrations 180 min after morphine administration were comparable among the three different treatments. TICM (1 mm) alone did not significantly affect the basal spinal GABA concentration for at least 240 min (fig. 3).
Back to Top | Article Outline
Behavioral Study
Effects of Intrathecal Bicuculline or TICM.
Table 1
Table 1
Image Tools
Table 2
Table 2
Image Tools
Intrathecal administration of bicuculline at doses of greater than 0.02 μg shortened the tail-flick latency in the tail-flick test. For example, 0.2 μg bicuculline shortened the tail-flick latency, which was observed within 5 min after the administration and continued for 30 min (table 1). Doses of intrathecal TICM greater than 1.0 μg also shortened the tail-flick latency. For example, 10.0 μg TICM shortened the tail-flick latency that was observed within 5 min and continued for 30 min (table 2). Neither 0.02 μg intrathecal bicuculline nor 1.0 μg TICM affected the basal tail-flick latency (tables 1 and 2). Therefore, these doses were used in the behavioral study, and the antagonistic effects of these drugs were assessed at 10 min after intrathecal administration.
Back to Top | Article Outline
Antagonistic Effects of Intrathecal Bicuculline and TICM on the Intracerebroventricular Morphine–induced Antinociception
Fig. 4
Fig. 4
Image Tools
Fig. 5
Fig. 5
Image Tools
The mean baseline tail-flick latency in this experiment was 5.7 s (range, 5.1–6.2 s). Figure 4 shows the time course of the change in percent maximum possible effect after intracerebroventricular morphine administration. Intracerebroventricular morphine (40 nmol) produced significant antinociceptive effects from 30 min up to 360 min after its administration. The maximal antinociceptive effect was observed 30–180 min after morphine administration. Figure 5 shows the effects of intrathecal bicuculline and TICM on the intracerebroventricular morphine–induced antinociceptive effect. We evaluated the antagonistic effects of bicuculline and TICM 180 min after morphine administration, because at this time point, the maximal effect of morphine in GABA analysis was observed (fig. 2). Intrathecal bicuculline significantly reversed the morphine-induced antinociception (P < 0.05). Intrathecal TICM also significantly reversed the morphine-induced antinociception (P < 0.05). On the other hand, intrathecal ACSF alone did not produce any significant changes in the tail-flick latency. There were no significant differences in percent maximum possible effect before intrathecal administration among the three different treatments.
Back to Top | Article Outline

Discussion

Intracerebroventricular Morphine–induced Spinal Serotonin Release
The current study showed that intracerebroventricular morphine evoked spinal 5-HT release. Previous reports also found morphine–induced spinal 5-HT release. 9–11 The major source of serotonergic projections to the dorsal horn are neurons in the rostral ventromedial medulla, which contains both opioid peptides and opioid receptors. 12,13 Several investigators found that electrical or chemical stimulation of the dorsal lateral funiculus, bulbospinal neurons, or the nucleus raphe magnus evoked 5-HT release in the dorsal horn of the spinal cord. 14–16 Therefore, it would be considered that morphine evokes spinal 5-HT release through activation of rostral ventromedial medulla serotonergic neurons by the inhibition of inhibitory interneurons. However, this may not be always consistent. First, Matos et al.17 concluded that intracerebroventricular morphine at an analgesic dose did not necessarily increase the extracellular concentration of 5-HT in the spinal dorsal horn using high-performance liquid chromatography with electrochemical detection, because morphine increased 5-HT release in only two of four rats. The discrepancy between their results and ours may be caused by the sensitivity of assay system for 5-HT. Baseline 5-HT concentrations were close to the sensitivity limit in the high-performance liquid chromatography with electrochemical detection, and hence it is difficult to measure small changes reliably. 18 We used a smaller diameter (2.1 mm) detector column in the current study, compared with that used in the study by Matos et al.17 (4.6 mm). This column enabled us to analyze 5-HT with high sensitivity. Using this assay system, morphine at the dose we used (40 nmol = 11.4 μg), which was almost comparable to the dose used by Matos et al. (10 μg), consistently increased spinal 5-HT concentration in all eight rats in the current study, and the degree of increase in 5-HT concentration was approximately 150% of the basal concentration. We performed the experiments 18–24 h after the implantation of the dialysis probe because of prevention of the decrease in recovery rate caused by gliosis. Therefore, there may be the possibility that the acute trauma of probe implantation modified the effect of morphine on 5-HT release. Second, electrophysiologic studies have demonstrated that most serotonergic rostral ventromedial medulla neurons are not affected by morphine. 19–21 However, those studies used the anesthetized animal. There is a possibility that anesthesia reduced serotonergic neuronal activity, 18,22 and serotonergic neuronal activity is lower during sleep than wakefulness. 23 We used the awake free-moving rats implanted with microdialysis probe in this study.
Back to Top | Article Outline
Intracerebroventricular Morphine–induced Activation of Spinal 5-HT3 Receptors and Spinal γ-Aminobutyric Acid Release
Several lines of study have suggested that supraspinal morphine or PAG stimulation activates a spinal GABAergic system. 1,3,5,24–26 However, there has not been direct evidence that supraspinal morphine evokes spinal GABA release. In the current study, we clearly demonstrated that intracerebroventricular morphine evokes spinal GABA release concomitant with 5-HT release. There was the discrepancy in time course between intracerebroventricular morphine–induced increases in 5-HT and GABA concentrations in our results. Spinal microdialysis does not directly measure neurotransmitter concentrations at synaptic sites, but rather neurotransmitter concentrations that have diffused into the extracellular space. Diffusion into the extracellular space is likely to involve processes of uptake and metabolism, which can modify the amount of neurotransmitter in the dialysate. Therefore, it is possible that diffusion into the extracellular space would contribute to the discrepancy in time course between 5-HT and GABA concentration.
There are at least four subtypes of 5-HT receptors (5-HT1, 5-HT2, 5-HT3, and 5-HT4) in the spinal cord. 27,28 Among the subtypes of 5-HT receptors, 5-HT3 receptors are unique because G proteins are not affected by agonist binding to 5-HT3 receptors. 5-HT3 receptors are directly linked to the opening of nonselective monovalent cation channels 4 and should mediate neuronal excitation. Alhaider et al.3 demonstrated that the activation of the GABAergic system contributed to spinal 5-HT3 receptor–mediated antinociception. Therefore, we focused on 5-HT3 receptors. TICM used in the current study is a potent and selective 5-HT3 receptor antagonist and has been commonly known as IC 205–930. Radioligands of IC 205–930 have been used to detect 5-HT3 receptor binding site in the central nervous system. 29 In the current study, blockade of spinal 5-HT3 receptors inhibited intracerebroventricular morphine–induced spinal GABA release. This indicates that morphine activates supraspinal serotonergic neurons projecting to the spinal cord, and then released 5-HT excites spinal GABAergic interneuron through 5-HT3 receptors. Immunohistochemical and autoradiographic studies revealed that 5-HT3 receptors are restricted to the superficial layers of the dorsal horn in the spinal cord. 29–31 Intense GABA immunoreactivities are also found within laminae I–III of the spinal cord. 32,33 It would be supposed that the GABAergic neuron with 5-HT3 receptors are directly activated or activation of presynaptic 5-HT3 receptors indirectly induce the activation of GABAergic neuron. There is a report that the 5-HT3 receptors mainly were located in the primary afferent terminal in the dorsal horn. 34 In addition, Peng et al.35 recently suggested that 5-HT3 receptors located in central terminals of primary afferents might contribute to the generation of dorsal root reflex, leading to presynaptic inhibition. Therefore, further study is necessary to clarify the mechanism of 5-HT3 receptor activation-induced GABA release.
Back to Top | Article Outline
Contributions of 5-HT3 Receptor Activation and γ-Aminobutyric Acid Release to Supraspinal Morphine–induced Antinociceptive Effect
In a behavioral study, we examined whether spinal GABA release through the activation of 5-HT3 receptors observed in our microdialysis study would be a component of supraspinal morphine–induced antinociception. GABA and 5-HT concentrations were measured within dorsal horn at the level of the L1 vertebra, and the tail-flick test was used in the behavioral study. The primary afferent neurons responsible for tail-flick response largely innervate in the dorsal horn of the sacrococcygeal spinal cord. In this study, GABA and 5-HT concentrations were measured within dorsal horn at the level of the L1 vertebra at which L4 nerve root mainly terminated. Therefore, results of the current microdialysis study may not directly reflect the changes of GABA and 5-HT in the sacral spinal cord. However, neurons originating in the nucleus raphe magnus, on which intracerebroventricular morphine acts, project to all levels of the spinal cord. 36–38 Therefore, intracerebroventricular morphine–induced GABA and 5-HT release we observed should contribute to antinociception in the tail-flick test.
It has been generally considered that supraspinal morphine produces antinociceptive effects through the serotonergic and noradrenergic descending inhibitory systems. In respect to the serotonergic mechanism, the analgesic action of morphine is reduced by destruction of the serotonergic innervation of the spinal cord with selective neurotoxins 39 or lesions of the medullary raphe, 40,41 which contains the cell of the origin of the bulbospinal 5-HT pathway innervating the spinal dorsal horn. Intrathecal injection of a 5-HT receptor antagonist also attenuates the analgesia produced by microinjection of morphine into the PAG. 42 In addition, intrathecal 5-HT produces antinociceptive effects. However, the nature of the receptors involved in the 5-HT–induced modulation of pain in the spinal cord remains to be elucidated. To the best of our knowledge, there has been no report that examined the contribution of spinal 5-HT3 receptors to intracerebroventricular morphine–induced antinociception in behavioral tests. The results of the current study have shown that spinal 5-HT3 receptors are involved in supraspinal morphine–induced antinociceptive effects. In contrast, several studies found only a minor role of the serotonergic system in supraspinal morphine–induced antinociceptive effect. 43–45 However, because these studies used methysergide as the 5-HT receptor antagonist, which blocks 5-HT1/2, not 5-HT3 receptors, the role of a serotonergic mechanism may be underestimated. The current study showed that TICM did not completely reverse the morphine-induced antinociceptive effect. This suggests the involvement of the other subtypes of 5-HT receptors or noradrenergic system in intracerebroventricular morphine–induced analgesia. Indeed, it has been reported that GABAA receptor response is potentiated by the activation of 5-HT2 receptor, 46 and that 5-HT1 receptor activation inhibits the nociceptive transmission. 47 The contribution of noradrenergic system in intracerebroventricular morphine–induced analgesia was also well demonstrated.
Several investigators reported that the spinal GABA system contributes to nucleus raphe magnus or PAG stimulation– and supraspinal morphine–induced antinociceptive effects. 1,3,5,25,26,48,49 Although our microdialysis study demonstrated that intracerebroventricular morphine gradually increased spinal GABA release with a maximal effect at 180–240 min after administration, an antinociceptive effect was observed 30 min after the administration and thereafter. Because we did not assess the antagonistic effect of bicuculline at the early phase of morphine-induced antinociception, the involvement of GABA in morphine analgesia at the early phase is unclear. We speculate that another mechanism such as noradrenergic or direct serotonergic inhibitions, in addition to GABAergic inhibition, might be involved in the antinociceptive effect observed in the early phase.
In conclusion, the current study showed that intracerebroventricular morphine produced antinociceptive effect, in part through GABA release by the activation of 5-HT3 receptors in the spinal cord.
Back to Top | Article Outline

References

1. Suh HW, Kim YH, Choi YS, Choi SR, Song DK: Effects of GABA receptor antagonists injected spinally on antinociception induced by opioids administered supraspinally in mice. Eur J Pharmacol 1996; 307: 141–7

2. Sweeney MI, White TD, Sawynok J: Intracerebroventricular morphine releases adenosine and adenosine 3′,5′-cyclic monophosphate from the spinal cord via a serotonergic mechanism. J Pharmacol Exp Ther 1991; 259: 1013–8

3. Alhaider AA, Lei SZ, Wilcox GL: Spinal 5-HT3 receptors-mediated antinociception: Possible release of GABA. J Neurosci 1991; 11: 1881–8

4. Derkach V, Surprenant A, North RA: 5-HT3 receptors menbrane ion channels. Nature 1989; 339: 706–9

5. Peng YB, Lin Q, Willis WD: The role of 5-HT3 receptors in periaqueductal gray-induced inhibition of nociceptive dorsal horn neurons in rats. J Pharmacol Exp Ther 1996; 276: 116–24

6. Bardin L, Lavarenne J, Eschalier A: Serotonin receptor subtypes involved in the spinal antinociceptive effect of 5-HT in rats. Pain 2000; 86: 11–8

7. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates, 2nd edition. Edited by Paxinos G, Watson C. Australia: Academic Press, 1986, pp 21–2

8. Skilling SR, Smullin DH, Beitz AJ, Larson AA: Extracellular amino acid concentrations in the dorsal spinal cord of freely moving rats following veratridine and nociceptive stimulation. J Neurochem 1988; 51: 127–32

9. Yaksh TL, Tyce GM: Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from spinal cord. Brain Res 1979; 171: 176–81

10. Bardon T, Ruckebusch: Changes in 5-HIAA and 5-HT levels in lumbar CSF following morphine administration to conscious dogs. Neurosci Lett 1984; 49: 147–51

11. Suzuki Y, Taguchi K: In vivo voltammetric studies of the effect of morphine on the serotonergic system in the cat spinal cord. Brain Res 1986; 398: 413–8

12. Oliveras JL, Bourgoin S, Hery F, Besson JM, Hamon M: The topographical distribution of serotoninergic terminals in the spinal cord of the cat: Biochemical mapping by the combined use of microdissection and microassay procedures. Brain Res 1977; 138: 393–406

13. Kwiat GC, Basbaum AI: The origin of brainstem noradrenergic and serotonergic projections to the spinal cord dorsal horn in the rat. Somatosens Mot Res 1992; 9: 157–73

14. Sorkin LS, McAdoo DJ, Willis WD: Raphe magnus stimulation-induced antinociception in the cat is associated with release of amino acids as well as serotonin in the lumbar dorsal horn. Brain Res 1993; 618: 95–108

15. Pilowsky PM, Kapoor V, Minson JB, West MJ, Chalmers JP: Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection. Brain Res 1986; 366: 354–7

16. Sorkin LS, Steinman JL, Hughes MG, Willis WD, McAdoo DJ: Microdialysis recovery of serotonin released in spinal cord dorsal horn. J Neurosci Methods 1988; 23: 131–8

17. Matos FF, Rollema H, Brown JL, Basbaum AI: Do opioids evoke the release of serotonin in the spinal cord? An in vivo microdialysis study of the regulation of extracellular serotonin in the rat. Pain 1992; 48: 439–47

18. Tao R, Auerbach SB: Anesthetics block morphine-induced increases in serotonin release in rat CNS. Synapse 1994; 18: 307–14

19. Gao K, Chen DO, Genzen JR, Mason P: Activation of serotonergic neurons in the raphe magnus is not necessary for morphine analgesia. J Neurosci 1998; 18: 1860–8

20. Potrebic SB, Fields HL, Mason P: Serotonin immunoreactivity is contained in one physiological cell class in the rat rostral ventromedial medulla. J Neurosci 1994; 14: 1655–65

21. Chiang CY, Pan ZZ: Differential responses of serotonergic and non-serotonergic neurons in nucleus raphe magnus to systemic morphine in rats. Brain Res 1985; 337: 146–50

22. Rivot JP, Pointis D, Besson JM: Morphine increases 5-HT metabolism in the nucleus raphe magnus: An in vivo study in freely moving rats using 5-hydroxyindole electrochemical detection. Brain Res 1988; 446: 333–42

23. Jacobs BL, Fornal CA, Wilkinson LO: Neurophysiological and neurochemical studies of brain serotonergic neurons in behaving animals. Ann N Y Acad Sci 1990; 600: 260–8

24. Xu TL, Pang, ZP, Li JS, Akaike N: 5-HT potentiate of the GABAA response in the rat sacral dorsal commissural neurons. Br J Pharmacol 1998; 124: 779–87

25. Rady JJ, Fujimoto JM: Supraspinal delta2 opioid agonist analgesia in swiss-webster mice involves spinal GABAA receptors. Pharmacol Biochem Behav 1996; 54: 363–9

26. Lin Q, Peng Y, Willis WD: Glycine and GABAA antagonists reduce the inhibition of primate spinothalamic tract neurons produced by stimulation in periaqueductal gray. Brain Res 1994; 654: 286–302

27. Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphery PP: International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol Rev 1994; 46: 157–203

28. Frazer A, Hensler JM: Serotonin, Basic Neurochemistry, 5th edition. Edited by Siegel GJ, Agranoff BW, Albers RW, Molinoff PB. New York, Raven, 1993, pp 283–308

29. Waeber C, Hoyer D, Palacios JM: 5-hydroxytryptamine3 receptors in the human brain: Autoradiographic visualization using [3H]ICS 205–930. Neuroscience 1989; 31: 393–400

30. Morales M, Battenberg E, Bloom FE: Distribution of neurons expressing immunoreactivity for the 5HT3 receptor subtype in the rat brain and spinal cord. J Comp Neurol 1998; 402: 385–401

31. Laporte AM, Koscielniak T, Ponchant M, Verge D, Hamon H, Gozlan H: Quantitative autoradiographic mapping of 5HT3 receptors in the rat CNS using[125I]iodo-zacopride and [3H]zacopride as radioligands. Synapse 1992; 10: 271–81

32. Magoul R, Onteniente B, Geffard M, Calas A: Anatomical distribution and ultrastructural organization of the GABAergic system in the rat spinal cord: An immunocytochemical study using anti-GABA antibodies. Neuroscience 1987; 20: 1001–9

33. Todd AJ, Mckenzie J: GABA-immunoreactive neurons in the dorsal horn of the rat spinal cord. Neuroscience 1989; 31: 799–806

34. Kidd EJ, Laporte AM, Langlois X, Fattaccini CM, Doyen C, Lombard MC, Gozlan H, Hamon M: 5-HT3 receptors in the rat central nervous system are mainly located on nerve fibres and terminals. Brain Res 1993; 612: 289–98

35. Peng YB, Wu J, Willis WD, Kenshalo DR: GABAA and 5-HT3 receptors are involved in dorsal root reflexes: Possible role in periaqueductal gray descending inhibition. J Neurophysiol 2001; 86: 49–58

36. Basbaum AI, Clanton CH, Fields HL: Three bulbospinal pathways from the rostral medulla of the cat: An autoradiographic study of pain modulating systems. J Comp Neurol 1978; 178: 209–24

37. Holstege G, Kuypers HG: The anatomy of brain stem pathways to the spinal cord in cat: A labeled amino acid tracing study. Prog Brain Res 1982; 57: 145–75

38. Martin RF, Haber LH, Willis WD: Primary afferent depolarization of identified cutaneous fibers following stimulation in medial brain stem. J Neurophysiol 1979; 42: 779–90

39. Vogt M: The effect of lowering the 5- hydroxytryptamine content of the rat spinal cord on analgesia produced by morphine. J Physiol 1974; 236: 483–98

40. Yaksh TL, Plant RL, Rudy TA: Studies on the antagonism by raphe lesions of the antinociceptive action of systemic morphine. Eur J Pharmacol 1977; 41: 399–408

41. Proudfit HK: Effects of raphe magnus and raphe pallidus lesions on morphine-induced analgesia and spinal cord monoamines. Pharmacol Biochem Behav 1980; 13: 705–14

42. Yaksh TL: Direst evidence that spinal serotonin and noradrenaline terminals mediate the spinal antinociceptive effects of morphine in the periaqueductal gray. Brain Res 1979; 160: 180–5

43. Tseng LL, Tang R: Differential actions of the blockade of spinal opioid, adrenergic and serotonergic receptors on the tail-flick inhibition induced by morphine microinjected into dorsal raphe and central gray in rats. Neuroscience 1989; 33: 93–100

44. DeLander GE, Wahl JJ: Morphine (intracerebroventricular) activates spinal systems to inhibit behavior induced by putative pain neurotransmitters. J Pharmacol Exp Ther 1989; 251: 1090–5

45. Wigdor S, Wilcox GL: Central and systemic morphine-induced antinociception in mice: Contribution of descending serotonergic and noradrenergic pathways. J Pharmacol Exp Ther 1987; 242: 90–5

46. Li H, Lang B, Kang JF, Li YQ: Serotonin potentiates the response of neurons of the superficial laminae of the rat spinal dorsal horn to gamma-aminobutyric acid. Brain Res Bull 2000; 52: 559–65

47. el-Yassir N, Fleetwood-Walker SM, Mitchell R: Heterogeneous effects of serotonin in the dorsal horn of rat: The involvement of 5-HT1 receptor subtypes. Brain Res 1988; 456: 147–58

48. McGowan MK, Hammond DL: Antinociception produced by microinjection of L-glutamate into the ventromedial medulla of the rat: mediation by spinal GABAA receptors. Brain Res 1993; 620: 86–96

49. Lin Q, Peng TB, Willis WD: Role of GABA receptor subtypes in inhibition of primate spinothalamic tract neurons: Difference between spinal and periaqueductal gray inhibition. J Neurophysiol 1996; 75: 109–23

Cited By:

This article has been cited 16 time(s).

Pharmacology Biochemistry and Behavior
Systemic administration of 5-HT2c receptor agonists attenuates muscular hyperalgesia in reserpine-induced myalgia model
Ogino, S; Nagakura, Y; Tsukamoto, M; Watabiki, T; Ozawa, T; Oe, T; Shimizu, Y; Ito, H
Pharmacology Biochemistry and Behavior, 108(): 8-15.
10.1016/j.pbb.2013.04.007
CrossRef
Experimental Neurology
The novel analgesic and high-efficacy 5-HT1A receptor agonist F 13640 inhibits nociceptive responses, wind-up, and after-discharges in spinal neurons and withdrawal reflexes
You, HJ; Colpaert, FC; Arendt-Nielsen, L
Experimental Neurology, 191(1): 174-183.
10.1016/j.expneurol.2004.08.031
CrossRef
Journal of Sexual Medicine
Successful Transcutaneous Electrical Nerve Stimulation in Two Women with Restless Genital Syndrome: The Role of A delta- and C-Nerve Fibers
Waldinger, MD; de Lint, GJ; Venema, PL; van Gils, APG; Schweitzer, DH
Journal of Sexual Medicine, 7(3): 1190-1199.
10.1111/j.1743-6109.2009.01578.x
CrossRef
Natural Product Reports
beta-phenylethylamines and the isoquinoline alkaloids
Bentley, KW
Natural Product Reports, 21(3): 395-424.
10.1039/b212259f
CrossRef
Brain Research
The activation of 5-HT3 receptors evokes GABA release in the spinal cord
Kawamata, T; Omote, K; Toriyabe, M; Yamamoto, H; Namiki, A
Brain Research, 978(): 250-255.
10.1016/S0006-8993(03)02952-4
CrossRef
Pain
Effects of systemic administration of lidocaine and QX-314 on hyperexcitability of spinal dorsal horn neurons after incision in the rat
Kawamata, M; Sugino, S; Narimatsu, E; Yamauchi, M; Kiya, T; Furuse, S; Namiki, A
Pain, 122(): 68-80.
10.1016/j.pain.2006.01.004
CrossRef
Neuroscience
Spinal pharmacology of antinociception produced by microinjection of mu or delta opioid receptor agonists in the ventromedial medulla of the rat
Hurley, RW; Banfor, P; Hammond, DL
Neuroscience, 118(3): 789-796.
10.1016/S0306-4522(03)00041-1
CrossRef
Brain Research
Release of GABA and activation of GABA(A) in the spinal cord mediates the effects of TENS in rats
Maedai, Y; Lisi, TL; Vance, CGT; Sluka, KA
Brain Research, 1136(1): 43-50.
10.1016/j.brainres.2006.11.061
CrossRef
Neuroscience Letters
5-HT3A receptor subunit is expressed in a subpopulation of GABAergic and enkephalinergic neurons in the mouse dorsal spinal cord
Huang, J; Wang, YY; Wang, W; Li, YQ; Tamamaki, N; Wu, SX
Neuroscience Letters, 441(1): 1-6.
10.1016/j.neulet.2008.04.105
CrossRef
International Journal of Neuroscience
Effect of GABA receptor agonists or antagonists on morphine-induced Straub tail in mice
Zarrindast, MR; Ghadimi, M; Ramezani-Tehrani, B; Sahebgharani, M
International Journal of Neuroscience, 116(8): 963-973.
10.1080/00207450600550428
CrossRef
British Journal of Pharmacology
Systemic morphine produce antinociception mediated by spinal 5-HT7, but not 5-HT1A and 5-HT2 receptors in the spinal cord
Dogrul, A; Seyrek, M
British Journal of Pharmacology, 149(5): 498-505.
10.1038/sj.bjp.0706854
CrossRef
Peptides
Endogenous opiates and behavior: 2002
Bodnar, RJ; Hadjimarkou, MM
Peptides, 24(8): 1241-1302.
10.1016/j.peptides.2003.08.002
CrossRef
European Neuropsychopharmacology
Intracerebroventricular injection of trazodone produces 5-HT receptor subtype mediated anti-nociception at the supraspinal and spinal levels
Zhang, RH; Nagata, T; Hayashi, T; Miyata, M; Kawakami, Y
European Neuropsychopharmacology, 14(5): 419-424.
10.1016/j.euroneuro.2003.12.006
CrossRef
Pain
Evidence for a monoamine mediated, opioid-independent, antihyperalgesic effect of venlafaxine, a non-tricyclic antidepressant, in a neurogenic pain model in rats
Marchand, F; Alloui, A; Chapuy, E; Jourdan, D; Pelissier, T; Ardid, D; Hernandez, A; Eschalier, A
Pain, 103(3): 229-235.
10.1016/S0304-3959(03)00168-4
CrossRef
Pain
Spinal 5-HT2 and 5-HT3 receptors mediate low, but not high, frequency TENS-induced antihyperalgesia in rats
Radhakrishnan, R; King, EW; Dickman, JK; Herold, CA; Johnston, NF; Spurgin, ML; Sluka, KA
Pain, 105(): 205-213.
10.1016/S0304-3959(03)00207-0
CrossRef
Progress in Neuro-Psychopharmacology & Biological Psychiatry
The antinociceptive effect of reversible monoamine oxidase-A inhibitors in a mouse neuropathic pain model
Villarinho, JG; Pinheiro, KD; Pinheiro, FD; Oliveira, SM; Machado, P; Martins, MAP; Bonacorso, HG; Zanatta, N; Fachinetto, R; Ferreira, J
Progress in Neuro-Psychopharmacology & Biological Psychiatry, 44(): 136-142.
10.1016/j.pnpbp.2013.02.005
CrossRef
Back to Top | Article Outline

© 2002 American Society of Anesthesiologists, Inc.

Publication of an advertisement in Anesthesiology Online does not constitute endorsement by the American Society of Anesthesiologists, Inc. or Lippincott Williams & Wilkins, Inc. of the product or service being advertised.
Login

Article Tools

Images

Share