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What are the effects of tramadol



Effects of Tramadol on Substantia Gelatinosa Neurons in the Rat

12.26.2018 | Alexander Mercer
What are the effects of tramadol

The effects of intravenous administration (0.5, 5, 15 mg/kg) of tramadol were evaluated. The effects of superfusion of tramadol on the surface of the spinal cord and of a tramadol metabolite (M1) were further analyzed. Intravenous administration of tramadol at doses >5 mg/kg decreased the sEPSCs and.

These inhibitory effects reached a maximum 5–15 min after the injections at all doses tested. Tramadol at a dose of 5 mg/kg had no effect on sEPSCs when naloxone 4 μg/kg was administered intravenously 5 min before tramadol ( Fig 3C and 3D ). Intravenous tramadol at doses of 0.5, 5, and 15 mg/kg inhibited sEPSCs in 2 out of 5, 3 out of 5, and 4 out of 5 neurons, respectively. The trace in Fig 3A shows that sEPSCs decreased following the intravenous injection of 5 mg/kg tramadol. Naloxone alone had no effect on EPSCs. We examined the effects of systemically administered tramadol on spontaneous EPSCs (sEPSCs). The cumulative event distribution curves obtained from the recordings in Fig 3A showed that systemic tramadol (5 mg/kg) shifted the curve toward a lengthening of the inter-event interval and a decreasing amplitude of the EPSCs ( Fig 3B ).

Abbreviations: T0.5, tramadol 0.5 mg/kg; T5, 5 mg/kg; T15, 15 mg/kg; N, naloxone https://doi.org/10.1371/journal.pone.0125147.g003. Tramadol inhibited sEPSCs at doses higher than 5 mg/kg (* p < 0.05). Systemic tramadol (5 mg/kg) had no effect on sEPSCs 5 minutes after the administration of naloxone (4 μg/kg). (F) Tramadol did not induce outward currents even at a high dosage (15 mg/kg). Neither naloxone (4 μg/kg) itself nor tramadol (5 mg/kg) after naloxone had any effect on sEPSCs. Data are the mean ± SEM. (A) Systemic tramadol (5 mg/kg) inhibited sEPSCs. Asterisks indicate a significant difference from the control (* p < 0.05) (paired t -test). (E) Summary of the effects of tramadol on sEPSCs. The ordinate represents synaptic charge transfers of sEPSCs as percentages of the control (before the administration of drugs). (D) Cumulative probability plots obtained from the trace in panel C showed that systemic tramadol did not change inter-event intervals and amplitude when naloxone was used as a pretreatment. (B) Cumulative probability plots obtained from the trace in panel A showed a prolongation of inter-event intervals and a decrease in amplitude induced by tramadol.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. : 2015 Yamasaki et al.

Drugs for intravenous administration were injected via the left femoral vein. The interval necessary for the test drug to reach the surface of the spinal cord was 5 seconds. The amounts of tramadol dissolved in Krebs solution were 10 and 100 μM. The amounts of systemically-administered tramadol were 0.5, 5, and 15 mg/kg. Drugs for spinal applications were diluted in Krebs solution and superfused onto the surface through inlet and outlet glass pipettes by exchanging solutions at a constant perfusion rate and temperature. The dosage of intravenous tramadol applied under in vivo conditions was determined based on clinically relevant doses. At the end of the experiments, the rats were euthanized by the administration of an overdose of pentobarbital.

The following drugs were purchased from Sigma Aldrich Japan (Tokyo, Japan): tramadol hydrochloride, M1, naloxone hydrochloride dihydrate, bicuculline, strychnine, and CNQX.

An enhancement of sIPSCs by intravenously administered tramadol was detected in 2 out of 5, 6 out of 9, and 5 out of 6 neurons at doses of 0.5 (T0.5), 5 (T5), and 15 (T15), respectively. The trace in Fig 4A shows an increase in sIPSCs after the administration of tramadol (5 mg/kg). We determined the effects of systemically administered tramadol on spontaneous IPSCs (sIPSCs) in SG neurons.

Paw withdrawal thresholds (PWT) were sequentially evaluated before (control) and after the intraperitoneal administration of normal saline (NS), tramadol 0.5 mg/kg (T 0.5), 5 mg/kg (T 5), and naloxone 4 μg/kg (T+N) in this sequential order to the same rats. https://doi.org/10.1371/journal.pone.0125147.g001. Asterisks indicate a significant difference from the control (* p < 0.05) (one-way analysis of variance and Dunnett’s post hoc test). Data are the mean ± SEM. The PWT was elevated with tramadol 5 mg/kg ( n = 6) and was returned to the control level by naloxone.

The mechanical withdrawal threshold was quantified for the right paw from an average of 5 Aesthesiometer trials. Control measurements were obtained using normal saline and then 0.5 mg/kg of tramadol, 5 mg/kg of tramadol, and 4 μg/kg of naloxone were administered intraperitoneally and sequentially to the same rat. Rats were placed onto a perforated metal mesh platform, and mechanical stimuli were delivered to the hindpaw using the Dynamic Plantar Aesthesiometer (37450, Ugo Basile, Comerio, Italy). This instrument, located under the platform, raised a straight metal filament, 0.5 mm in diameter, until it contacted with the plantar surface of the hindpaw and exerted a gradually increasing upward force (from 1 to 50 g over 20 seconds) until the paw was withdrawn. Six male rats, aged 6 weeks, were included in this protocol. The rats were allowed to acclimate to the test location for 1 hour before the experiment. Pain thresholds were evaluated twenty minutes after the administration of tramadol or normal saline, and ten minutes after the administration of naloxone. Behavioral testing was conducted in a silent room away from the colony room, in daylight at a standard temperature (24 ± 1°C).

Competing interests: The authors have declared that no competing interests exist.

A previous study using microdialysis reported systemic tramadol-induced increases in noradrenaline and 5-HT in the spinal dorsal horn, indicating the involvement of descending inhibitory pathways in its antinociceptive mechanisms. These findings demonstrated the importance of understanding how systemic tramadol modulates synaptic transmission at SG neurons in the spinal cord in vivo. M1 has higher affinity and efficacy for opioid μ receptors than tramadol. Opioid receptors are located abundantly in pain pathways and in the descending inhibitory system. A previous electrophysiological study using rat spinal cord slices demonstrated that M1 induced outward currents by activating μ-receptors in SG neurons, which suggested that the marked hyperpolarization of SG neurons may inhibit nociception. Tramadol and its metabolite O-desmethyl tramadol (M1) have the opioid analgesic properties.

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The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This study was supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science, Tokyo, Japan.

In the present study, we used the in vivo patch-clamp technique to record spontaneous excitatory post synaptic currents (sEPSCs), spontaneous inhibitory post synaptic currents (sIPSCs), and slow membrane currents from SG neurons in the rat spinal dorsal horn.

Academic Editor: Theodore John Price, University of Texas at Dallas, UNITED STATES.

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Affiliation Department of Anesthesiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

In all cases, n referred to the number of neurons tested. Significantly different values were indicated by asterisks in the figures or by the Kolmogorov–Smirnov test for their probabilities. Modification of areas of sEPSCs and sIPSCs were assessed as the percentage of control. Sample size calculation was based on the values of synaptic charge transfer in our previous study. We defined neurons as being sensitive to a particular drug when the synaptic charge transfer was altered by more than ± 15% from the control. Significance was defined as p < 0.05 by a one-way analysis of variance (ANOVA) with Dunnett’s post hoc test for withdrawal threshold data and the paired t -test for the absolute values of the amplitude, frequency, or synaptic charge transfer of IPSCs and EPSCs. Five cells were required for each experiment (α = 0.05 and β = 0.2). All numerical data were expressed as the mean ± SEM. The total area of IPSCs and EPSCs over a 10 second period was measured to reflect the ongoing synaptic charge transfer, which indicated the intensity of each synaptic current.

Whole-cell voltage-clamp recordings were conducted from SG neurons with a patch electrode with a tip resistance of 8–12 MΩ and filled with a pipette solution containing the following (in mM): 110 Cs 2 SO 4, 5 TEA-Cl, 0.5 CaCl 2, 2 MgCl 2, 5 EGTA, 5 HEPES, and 5 Mg-ATP, in the 0mV voltage-clamp mode to observe GABAergic and/or glycinergic IPSCs, or 136 K-gluconate, 5 KCl, 0.5 CaCl 2, 2 MgCl 2, 5 EGTA, 5 HEPES, and 5 Mg-ATP, mainly in the -70 mV voltage clamp mode to observe EPSCs and slow membrane currents.

Received: December 18, 2014; Accepted: March 18, 2015; Published: May 1, 2015.

Systemic tramadol (5 mg/kg) had no effect on sIPSCs 5 minutes after the administration of naloxone (4 μg/kg). Abbreviations: T0.5, tramadol 0.5 mg/kg; T5, 5 mg/kg; T15, 15 mg/kg; N, naloxone https://doi.org/10.1371/journal.pone.0125147.g004. (E) Summary of tramadol effects on sIPSCs. Asterisks indicate a significant difference from the control (* p < 0.05) (paired t -test). (D) Cumulative probability plots obtained from the trace in panel C showed that systemic tramadol did not change inter-event intervals and amplitude when naloxone was used as a pretreatment. (A) Systemic tramadol (5 mg/kg) increased sIPSCs. Data are the mean ± SEM. Tramadol increased sIPSCs at doses higher than 5 mg/kg (* p < 0.05). The ordinate represents synaptic charge transfers of sIPSCs as percentages of the control (before the administration of drugs). (B) Cumulative probability plots obtained from the trace in panel A showed the shortening of inter-event intervals and increase in amplitude induced by tramadol. Neither naloxone (4 μg/kg) itself nor tramadol 5 mg/kg after naloxone had any effect on sIPSCs.

Affiliation Department of Anesthesiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

The amplitude of each postsynaptic current was defined as the amount from the initial inflection point (not from the baseline) to the peak. The frequencies, amplitudes, and synaptic charge transfers of spontaneous IPSCs (sIPSCs) and EPSCs (sEPSCs) were measured automatically using MiniAnalysis software (Synaptosoft, Decatur, GA). Data were digitized using an analog-to-digital converter (Digidata1321A, Molecular Devices, Union City, CA), stored on a personal computer using a data acquisition program (Clampex version 8.0; Molecular Devices, Union City, CA), and analyzed using a special software package (Clampfit version 4.1; Molecular Devices, Union City, CA). Signals were collected with a patch clamp amplifier (Axopatch 200B; Molecular Devices, Union City, CA). The validity of this method was confirmed by a visual analysis of all traces on a fast-time scale before they were accepted for further investigations. SG neurons were recorded at a depth of 50–150 μm from the dorsal surface of the spinal cord at the L2-L4 level.

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The pretreatment with systemic naloxone abolished tramadol-induced increases in sIPSCs (97 ± 8%, n = 5) ( Fig 4E ). The inhibitory synaptic charge transfers of sIPSCs were significantly enhanced by systemic tramadol at doses of 5 and 15 mg/kg. Fig 4C and 4D show that 5 mg/kg tramadol had no effect on spontaneous IPSCs when naloxone 4 μg/kg was administered intravenously 5 min before tramadol (N+T5). The enhancement observed in sIPSCs following systemic tramadol administration reached a maximum approximay 5–10 min after the injection. Tramadol shifted the cumulative event distributions of sIPSCs to shorten the inter-event interval and increase their amplitude ( Fig 4B ). These enhancements were 114 ± 9% ( n = 5), 129 ± 10% ( n = 9), and 157 ± 18% ( n = 6) of the control at doses of 0.5, 5, and 15 mg/kg, respectively ( Fig 4E ). Naloxone alone had no effect on sIPSCs.

Affiliation Department of Anesthesiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

Spinalized rats underwent a laminectomy at the cervical level to allow right-sided cord hemisection to disturb descending inhibitory pathways ipsilateral to the recording side. The lumbar spinal cord was exposed by thoracolumbar laminectomy at the level from Th13 to L2, and the rat was then placed in a stereotaxic apparatus (Model ST-7; Narishige, Tokyo, Japan). The experimental method was previously described at length. The pia-arachnoid membrane was cut to allow the patch electrode to enter the spinal cord. The surface of the spinal cord was irrigated with 95% O 2 -5% CO 2 -equilibrated Krebs solution (15 ml/min; NaCl, 117; KCl, 3.6; CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; glucose, 11; NaHCO3, 25 mM) through glass pipettes at 36.5°C. Briefly, 96 male Sprague-Dawley rats (6–9 weeks of age, 210–380 g) were anesthetized with urethane (1.2–1.5 g/kg intraperitoneally), and the left femoral vein was cannulated for drug administration. The dura was opened and then the dorsal root that enters the spinal cord above the level of the recording sites was lifted so that a recording electrode could be inserted into the SG from the surface of the spinal cord.

The effects of intravenous administration (0.5, 5, 15 mg/kg) of tramadol were evaluated. These effects were not observed following naloxone pretreatment. We used an in vivo patch clamp technique in urethane-anesthetized rats to determine the antinociceptive mechanism of tramadol. Intravenous administration of tramadol at doses >5 mg/kg decreased the sEPSCs and increased the sIPSCs in SG neurons. The present study demonstrated that systemically administered tramadol indirectly inhibited glutamatergic transmission, and enhanced GABAergic and glycinergic transmissions in SG neurons. These effects were mediated primarily by the activation of μ-opioid receptors. Tramadol is thought to modulate synaptic transmissions in the spinal dorsal horn mainly by activating µ-opioid receptors and by inhibiting the reuptake of monoamines in the CNS. In vivo whole-cell recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) were made from substantia gelatinosa (SG) neurons (lamina II) at holding potentials of 0 mV and -70 mV, respectively. The effects of superfusion of tramadol on the surface of the spinal cord and of a tramadol metabolite (M1) were further analyzed. M1 may play a key role in the antinociceptive mechanisms of tramadol. Tramadol superfusion at a clinically relevant concentration (10 µM) had no effect, but when administered at a very high concentration (100 µM), tramadol decreased sEPSCs, produced outward currents, and enhanced sIPSCs. However, the precise mode of modulation remains unclear. The effects of M1 (1, 5 mg/kg intravenously) on sEPSCs and sIPSCs were similar to those of tramadol at a corresponding dose (5, 15 mg/kg).

Affiliation Department of Anesthesiology, Osaka City University Graduate School of Medicine, Osaka, Japan *

The synaptic charge transfer of sIPSCs was 52 ± 11% of the control in the presence of strychnine and was further reduced to 5 ± 3% in the presence of bicuculline and strychnine (n = 5, p < 0.05). The synaptic charge transfer of sEPSCs was 4 ± 2% of the control in the presence of 10 μM CNQX ( n = 5, p < 0.05). These observations indicated that the sEPSCs were mostly mediated via AMPA/Kainate receptors, while the sIPSCs were mediated by both GABA A and glycine receptors. We confirmed the characteristics of sEPSCs and sIPSCs by superfusion of CNQX (an AMPA/Kainate antagonist), bicuculline (a GABA A receptor antagonist), and strychnine (a glycine receptor antagonist) onto the spinal cord. Superfusion of 20 μM bicuculline decreased the sIPSCs and coadministration of 2 μM strychnine with bicuculline almost compley eliminated them ( Fig 2C ). The sEPSCs rapidly disappeared almost compley after 10 μM CNQX superfusion and then recovered following washout of the solution ( Fig 2B ).

The outward currents were analyzed under the same conditions as those used for the EPSCs. The effects of tramadol were determined on spontaneous EPSCs (sEPSCs) and spontaneous IPSCs (sIPSCs) since the spontaneous currents were considered to represent synaptic activity of SG neurons. In SG neurons, the EPSCs and the IPSCs exhibited stable activity without noxious stimuli. These were so-called spontaneous currents. As this technique is blind patch-clamp recording, the sensory SG neurons were identified by the response of the EPSCs and IPSCs to a touch and pinch stimuli applied to rat hindpaw ( Fig 2A ). We used a previously described in vivo patch-clamp technique for successful recording of EPSCs and IPSCs from spinal dorsal horn neurons. The sEPSCs and sIPSCs exhibited sufficient amplitudes for evaluations at holding potentials of -70 mV and 0 mV, respectively, using each specific pipette solution.

Affiliation Department of Anesthesiology, Osaka City University Graduate School of Medicine, Osaka, Japan.

Citation: Yamasaki H, Funai Y, Funao T, Mori T, Nishikawa K (2015) Effects of Tramadol on Substantia Gelatinosa Neurons in the Rat Spinal Cord: An In Vivo Patch-Clamp Analysis. PLoS ONE 10(5): e0125147. https://doi.org/10.1371/journal.pone.0125147.

The superficial dorsal horn, specifically the substantia gelatinosa (SG) lamina II of the spinal cord, is involved in transmission of peripheral pain signals to the central nociceptive field. SG neurons receive noxious information by glutamatergic synaptic inputs from peripheral Aδ and C-afferent fibers. A recently developed “ in vivo patch-clamp” technique for the spinal dorsal horn has enabled the assessment of the actions of systemically administered drugs on synaptic activity in SG neurons. They also receive abundant inhibitory synaptic inputs from GABAergic and glycinergic interneurons, thus, they are modulated by the descending inhibitory system.

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Tramadol is widely used as an analgesic for the treatment of postoperative, cancer, or chronic neuropathic pain. Its analgesic effects have been reported following its systemic administration in rat acute and chronic pain models. Tramadol itself acts on several ion channels and receptors, including sodium channels, GABA A receptors, NMDA receptors, and nicotinic acetylcholine receptors. These findings suggest that sensory nociceptive transmission in the spinal cord may be modulated in several different ways. Two main mechanisms are thought to contribute to the antinociceptive action of tramadol in the central nervous system and spinal cord: the activation of μ opioid receptors and the inhibition of the neuronal uptake of noradrenaline and serotonin (5-HT).

Data Availability: All relevant data are within the paper.

(A) The identification of SG neurons was made by the response of the EPSCs and IPSCs to a pinch stimuli applied to rat hindpaw. (B) The perfusion of CNQX (10 μM) on the spinal cord immediay abolished spontaneous excitatory postsynaptic currents (sEPSCs). Coadministration of bicuculline (20 μM) and strychnine (2 μM) in the perfusion solution diminished spontaneous inhibitory postsynaptic currents (sIPSCs). https://doi.org/10.1371/journal.pone.0125147.g002.

All experimental procedures were approved by the Ethics Committee on Animal Experiments at Osaka City University (approval number: 13044) and performed according to the Guiding Principles for the Care and Use of Animals recommended by the Physiological Society of Japan. All efforts were made to minimize the number of animals used in the experiments.

The pretreatment of systemic naloxone abolished tramadol-induced decreases in EPSCs (N+T5) (96 ± 9%, n = 5) ( Fig 3E ). Obvious outward currents were not observed after intravenous tramadol even at a high dose ( Fig 3F ). A small dose of intravenous tramadol (0.5 mg/kg) did not significantly decrease sEPSCs (T0.5) (95 ± 8%, n = 5). We quantified the effects of tramadol on EPSCs by obtaining the synaptic charge transfers of EPSCs by calculating the area under the EPSCs for a 10-sec period and normalizing to the level before drug administrations (control). Tramadol at doses over 5 mg/kg decreased the synaptic charge transfer of sEPSCs to 79 ± 6% of the control (n = 5) at 5 mg/kg (T5) and 73 ± 6% ( n = 5) at 15 mg/kg (T15).