Analyses of the Mode of Action of an Alpha-Adrenoceptor Blocker in Substantia Gelatinosa Neurons in Rats.

To elucidate why naftopidil increases the frequency of spontaneous synaptic currents in only some substantia gelatinosa (SG) neurons, post-hoc analyses were performed. Blind patch-clamp recording was performed using slice preparations of SG neurons from the spinal cords of adult rats. Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were recorded. The ratios of the frequency and amplitude of the sIPSCs and sEPSCs following the introduction of naftopidil compared with baseline, and after the application of naftopidil, serotonin (5-HT), and prazosin, compared with noradrenaline (NA) were evaluated. First, the sIPSC analysis indicated that SG neurons reached their full response ratio for NA at 50 µM. Second, they responded to 5-HT (50 µM) with a response ratio similar to that for NA, but prazosin (10 µM) did not change the sEPSCs and sIPSCs. Third, the highest concentration of naftopidil (100 µM) led to two types of response in the SG neurons, which corresponded with the reactions to 5-HT and prazosin. These results indicate that not all neurons were necessarily activated by naftopidil, and that the micturition reflex may be regulated in a sophisticated manner by inhibitory mechanisms in these interneurons.

Antinociceptive effect of selective G protein-gated inwardly rectifying K+ channel agonist ML297 in the rat spinal cord.

The G protein-gated inwardly rectifying K+ (GIRK) channels play important signaling roles in the central and peripheral nervous systems. However, the role of GIRK channel activation in pain signaling remains unknown mainly due to the lack of potent and selective GIRK channel activators until recently. The present study was designed to determine the effects and mechanisms of ML297, a selective GIRK1/2 activator, on nociception in the spinal cord by using behavioral studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons. Rats were prepared for chronic lumber catheterization and intrathecal administration of ML297. The nociceptive flexion reflex was tested using an analgesy-meter, and the influence on motor performance was assessed using an accelerating rotarod. We also investigated pre- and post-synaptic actions of ML297 in spinal cord preparations by whole-cell patch-clamp recordings. Intrathecal administration of ML297 increased the mechanical nociceptive threshold without impairing motor function. In voltage-clamp mode of patch-clamp recordings, bath application of ML297 induced outward currents in a dose-dependent manner. The ML297-induced currents demonstrated specific equilibrium potential like other families of potassium channels. At high concentration, ML297 depressed miniature excitatory postsynaptic currents (mEPSCs) but not their amplitude. The ML297-induced outward currents and suppression of mEPSCs were not inhibited by naloxone, a µ-opioid receptor antagonist. These results demonstrated that intrathecal ML297 showed the antinociceptive effect, which was mediated through direct activation of pre- and post-synaptic GIRK channels. Selective GIRK channel activation is a promising strategy for the development of new agents against chronic pain and opioid tolerance.

Spinal CCL2 Promotes Pain Sensitization by Rapid Enhancement of NMDA-Induced Currents Through the ERK-GluN2B Pathway in Mouse Lamina II Neurons.

Previous studies have shown that CCL2 (C-C motif chemokine ligand 2) induces chronic pain, but the exact mechanisms are still unknown. Here, we established models to explore the potential mechanisms. Behavioral experiments revealed that an antagonist of extracellular signal-regulated kinase (ERK) inhibited not only CCL2-induced inflammatory pain, but also pain responses induced by complete Freund's adjuvant. We posed the question of the intracellular signaling cascade involved. Subsequent experiments showed that CCL2 up-regulated the expression of phosphorylated ERK (pERK) and N-methyl D-aspartate receptor [NMDAR] subtype 2B (GluN2B); meanwhile, antagonists of CCR2 and ERK effectively reversed these phenomena. Whole-cell patch-clamp recordings revealed that CCL2 enhanced the NMDAR-induced currents via activating the pERK pathway, which was blocked by antagonists of GluN2B and ERK. In summary, we demonstrate that CCL2 directly interacts with CCR2 to enhance NMDAR-induced currents, eventually leading to inflammatory pain mainly through the CCL2-CCR2-pERK-GluN2B pathway.

Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo.

The ability to detect noxious stimulation is essential to an organism's survival and wellbeing. Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem. The spinal cord dorsal horn is critically involved in nociception and chronic pain. Especially, the substantia gelatinosa (SG) neurons of lamina II, which receives nociceptive inputs from primary afferents. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood. Here, we studied synaptic plasticity on excitatory transmissions in the adult rat SG neurons. Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection. In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. The frequency and amplitude of sEPSCs in the nerve injury and the CFA group were reduced by the presence of tetrodotoxin (TTX). By contrast, TTX did not reduce the sEPSCs compared with miniature EPSCs in naïve rats. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group. Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

Comparison of ex vivo and in vitro actions of gabapentin in superficial dorsal horn and the role of extra-spinal sites of drug action.

Although gabapentin (GBP) is a first-line treatment in the management of neuropathic pain, its mechanism of action is incompletely understood. We have previously shown, in rats made neuropathic following sciatic chronic constriction injury, that IP injection of 100 mg/kg GBP decreases overall excitability of spinal cord slices obtained ex vivo. Excitability was assessed using confocal imaging to monitor the amplitude of K+- induced increases in cytoplasmic Ca2+. This decrease in excitability involved a reduction in the frequency and amplitude of spontaneous EPSC's (sEPSC) in putative excitatory substantia gelatinosa neurons and an increase in sEPSC frequency in putative inhibitory neurons. We used have whole-cell recording to compare these ex vivo actions of GBP with its acute in vitro effects on spinal cord slices obtained from neuropathic but drug-free rats. While GBP (100µM) decreased sEPSC amplitude and frequency in excitatory neurons in vitro in a similar fashion to effects observed ex vivo, sEPSC frequency in inhibitory neurons was decreased in vitro rather than increased. Acute in vitro application of GBP also failed to decrease the overall excitability of slices from neuropathic animals as monitored by confocal Ca2+ imaging. Since spinal cord slices in vitro are disconnected from the periphery and higher brain centres, the GBP-induced increase in sEPSC frequency in inhibitory neurons previously reported and seen ex vivo must result from extra-spinal actions. It may be attributable to alterations in descending neurotrophic control of dorsal horn circuitry.

Ethanol-induced enhancement of inhibitory synaptic transmission in the rat spinal substantia gelatinosa.

Recent studies have shown that ethanol produces a widespread modulation of neuronal activity in the central nervous system. It is not fully understood, however, how ethanol changes nociceptive transmission. We investigated acute effects of ethanol on synaptic transmission in the substantia gelatinosa (lamina II of the spinal dorsal horn) and mechanical responses in the spinal dorsal horn. In substantia gelatinosa neurons, bath application of ethanol at low concentration (10 mM) did not change the frequency and amplitude of spontaneous inhibitory postsynaptic currents. At medium to high concentrations (20-100 mM), however, ethanol elicited a barrage of large amplitude spontaneous inhibitory postsynaptic currents. In the presence of tetrodotoxin, such enhancement of spontaneous inhibitory postsynaptic currents was not detected. In addition, ethanol (20-100 mM) increased the frequency of spontaneous discharge of vesicular GABA transporter-Venus-labeled neurons and suppressed the mechanical nociceptive response in wide-dynamic range neurons in the spinal dorsal horn. The present results suggest that ethanol may reduce nociceptive information transfer in the spinal dorsal horn by enhancement of inhibitory GABAergic and glycinergic synaptic transmission.

The histology, physiology, neurochemistry and circuitry of the substantia gelatinosa Rolandi (lamina II) in mammalian spinal cord.

The substantia gelatinosa Rolandi (SGR) was first described about two centuries ago. In the following decades an enormous amount of information has permitted us to understand - at least in part - its role in the initial processing of pain and itch. Here, I will first provide a comprehensive picture of the histology, physiology, and neurochemistry of the normal SGR. Then, I will analytically discuss the SGR circuits that have been directly demonstrated or deductively envisaged in the course of the intensive research on this area of the spinal cord, with particular emphasis on the pathways connecting the primary afferent fibers and the intrinsic neurons. The perspective existence of neurochemically-defined sets of primary afferent neurons giving rise to these circuits will be also discussed, with the proposition that a cross-talk between different subsets of peptidergic fibers may be the structural and functional substrate of additional gating mechanisms in SGR. Finally, I highlight the role played by slow acting high molecular weight modulators in these gating mechanisms.

Nefopam downregulates autophagy and c-Jun N-terminal kinase activity in the regulation of neuropathic pain development following spinal nerve ligation.

BACKGROUND: Neurodegeneration is associated with changes in basal cellular function due to the dysregulation of autophagy. A recent study introduced the involvement of autophagy during spinal nerve ligation (SNL). Nefopam has shown potential for reducing neuropathic pain, but the underlying mechanisms are unknown. Here, we investigated the effects of nefopam on neuropathic pain development following SNL, focusing on the involvement of autophagy. METHODS: The functional role of nefopam in capsaicin-induced autophagy was assessed by human glioblastoma M059 K cells. The neuropathic pain model was used to determine whether the effect of nefopam on pain control was mediated through autophagy control. Neuropathic pain was induced by L5 and L6 SNL in male rats randomized into three groups: Group S (sham-operated), Group C (received normal saline), and Group E (received nefopam). A behavioral test using a von Frey was examined. Expression changes of autophagy in response to nefopam was analyzed in spinal cord tissues (L4-L6) by immunoblotting and immunohistochemistry. RESULTS: The paw withdrawal threshold examined on days 3, 5, 7, and 14 post-SNL was significantly higher in Group E than in Group C. SNL increased the levels of microtubule-associated protein 1 light chain 3B (LC3B-1), with concomitant reduction of sequestosome 1 (SQTSM1/p62), compared with Group S, indicating that SNL induced autophagy. These effects were reversed by nefopam injection, and the results were confirmed by immunohistochemistry for LC3-I/II. Furthermore, SNL-mediated JNK activation was markedly decreased following nefopam injection. Hematoxylin and eosin staining on Day 14 post-SNL revealed that SNL caused lymphocyte infiltration and oligodendrocyte localization in the substantia gelatinosa of the dorsal gray horn, which were reduced by nefopam injection. CONCLUSION: Collectively, the mode of action of nefopam on neuropathic pain appears to be associated with downregulation of phospho-JNK and autophagy, as well as modulation of the immune response.

Modulation by orexin A of spontaneous excitatory and inhibitory transmission in adult rat spinal substantia gelatinosa neurons.

Hypothalamic neuropeptides, orexins A and B, differently inhibit nociceptive behavior. This difference is possibly due to a distinction between orexins A and B in modulating synaptic transmission in spinal substantia gelatinosa (SG) neurons that play a pivotal role in regulating nociceptive transmission. Although we previously reported a modulatory action of orexin B on synaptic transmission in adult rat SG neurons, it has not been fully examined how the transmission is affected by orexin A. The present study examined the effects of orexin A on spontaneous excitatory and inhibitory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. Like orexin B, orexin A produced an inward current at -70 mV and/or increased the frequency of spontaneous excitatory postsynaptic current without changing its amplitude. Half-maximal effective concentration values for their effects were 0.0045 and 0.030 µM, respectively; the former value was four-fold smaller than that of orexin B while the latter value was comparable to that of orexin B. Orexin A enhanced not only glycinergic but also GABAergic transmission, although only glycinergic transmission was facilitated by orexin B in the majority of neurons tested. Orexin A activities were inhibited by an orexin-1 receptor antagonist (SB334867) but not an orexin-2 receptor antagonist (JNJ10397049), as different from orexin B whose activation was depressed by JNJ10397049 but not SB334867. These results indicate that orexin A has a different action from orexin B in SG neurons in efficacy for inward current production and in GABAergic transmission enhancement, possibly owing to orexin-1 but not orexin-2 receptor activation. This difference could contribute to at least a part of the distinction between orexins A and B in antinociceptive effects.

Characterization of Superficial Dorsal Horn Neurons from "Tamamaki" Mice and Stability of their GAD67-EGFP Phenotype in Defined-Medium Organotypic Culture.

Defined medium organotypic cultures (DMOTC) containing spinal dorsal horn neurons are especially useful in studying the etiology and pharmacology of chronic pain. We made whole-cell recordings from neurons in acutely isolated mouse spinal cord slices or from those maintained in DMOTC for up to 6 weeks. In acute slices, neurons in the substantia gelatinosa exhibited 7 different firing patterns in response to 800-ms depolarizing current commands; delay (irregular), delay (tonic), tonic, regular firing, phasic, initial bursting and single spiking. Initial bursting and regular firing neurons are not found in rat substantia gelatinosa. In acute slices from "Tamamaki" mice that express enhanced green fluorescent protein (EGFP) under the control of the glutamic acid decarboxylase 67 (GAD67) promotor, tonic, phasic and regular firing neurons exhibited the strongest GABAergic (GAD67-EGFP+) phenotype. Delay (tonic) and delay (irregular) neurons almost never expressed GAD67 (GAD67-EGFP-) and are likely glutamatergic. All seven phenotypes were preserved in mouse spinal cord neurons in DMOTC prepared from e12 embryos and the GAD67-EGFP+ phenotype continued to associate with phasic and regular firing neurons. Only 3 out of 51 GAD67-EGFP+ neurons exhibited a delay (tonic) firing pattern. Modifications to the mouse genome thus continue to be expressed when embryonic neurons develop in vitro in DMOTC. However, analysis of the amplitude and interevent interval of spontaneous EPSCs (sEPSCs) indicated substantial re-arrangement of synaptic connections within the cultures. Despite this, the characteristics and age-dependence of asynchronous oscillatory activity, as monitored by multiphoton Ca2+ imaging, were similar in acute slices and in DMOTC.

Reductions in tonic GABAergic current in substantia gelatinosa neurons and GABAA receptor δ subunit expression after chronic constriction injury of the sciatic nerve in mice.

BACKGROUND: Decreased Gamma-aminobutyric acid (GABA)-ergic phasic inhibitory transmission in the spinal cord is thought to be responsible for the development of neuropathic pain. However, the role of GABAergic tonic current in substantia gelatinosa (SG) neurons in neuropathic pain remains to be fully elucidated. In this study, we assessed GABAergic tonic currents of SG neurons in a sciatic nerve chronic constriction injury (CCI) mouse. METHOD: Whole-cell patch clamp recordings form lumbar spinal cord slices was performed to evaluate GABAergic currents. We also investigated the expression changes of GABAA receptor subunits which are considered to mediate tonic currents. RESULTS: The percentage of SG neurons receiving GABAergic tonic currents decreased in CCI mice compared with Naïve mice. No significant change was observed in the mean amplitude of GABAergic tonic currents. RT-PCR and Western blot revealed that the expression of GABAA receptor δ subunits decreased following CCI. CONCLUSION: A reduction in the expression the δ subunit of the GABAA receptor and diminished GABAergic tonic current in SG neurons were observed after CCI in mice. GABAergic tonic current plays a key role in neuropathic pain. The GABAA receptor δ subunit may be a therapeutic target in neuropathic pain. WHAT DOES THIS STUDY ADD?: In spinal SG neurons, GABAergic inhibitory transmission operates through both phasic and tonic currents, but physiological role is largely unknown. In this study, we report dysregulation of GABAA receptor δ subunit-mediated tonic current in SG neurons may result in spinal disinhibition resulting in neuropathic pain in CCI mice.

The Gate Theory of Pain Revisited: Modeling Different Pain Conditions with a Parsimonious Neurocomputational Model.

The gate control theory of pain proposed by Melzack and Wall in 1965 is revisited through two mechanisms of neuronal regulation: NMDA synaptic plasticity and intrinsic plasticity. The Melzack and Wall circuit was slightly modified by using strictly excitatory nociceptive afferents (in the original arrangement, nociceptive afferents were considered excitatory when they project to central transmission neurons and inhibitory when projecting to substantia gelatinosa). The results of our neurocomputational model are consistent with biological ones in that nociceptive signals are blocked on their way to the brain every time a tactile stimulus is given at the same locus where the pain was produced. In the computational model, the whole set of parameters, independently of their initialization, always converge to the correct values to allow the correct computation of the circuit. To test the model, other painful conditions were analyzed: phantom limb pain, wind-up and wind-down pain, breakthrough pain, and demyelinating syndromes like Guillain-Barré and multiple sclerosis.

Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons.

BACKGROUND: Lidocaine, which blocks voltage-gated sodium channels, is widely used in surgical anesthesia and pain management. Recently, it has been proposed that the hyperpolarization-activated cyclic nucleotide (HCN) channel is one of the other novel targets of lidocaine. Substantia gelatinosa in the spinal dorsal horn, which plays key roles in modulating nociceptive information from primary afferents, comprises heterogeneous interneurons that can be electrophysiologically categorized by firing pattern. Our previous study demonstrated that a substantial proportion of substantia gelatinosa neurons reveal the presence of HCN current (Ih); however, the roles of lidocaine and HCN channel expression in different types of substantia gelatinosa neurons remain unclear. METHODS: By using the whole-cell patch-clamp technique, we investigated the effect of lidocaine on Ih in rat substantia gelatinosa neurons of acute dissociated spinal cord slices. RESULTS: We found that lidocaine rapidly decreased the peak Ih amplitude with an IC50 of 80 µM. The inhibition rate on Ih was not significantly different with a second application of lidocaine in the same neuron. Tetrodotoxin, a sodium channel blocker, did not affect lidocaine's effect on Ih. In addition, lidocaine shifted the half-activation potential of Ih from -109.7 to -114.9 mV and slowed activation. Moreover, the reversal potential of Ih was shifted by -7.5 mV by lidocaine. In the current clamp, lidocaine decreased the resting membrane potential, increased membrane resistance, delayed rebound depolarization latency, and reduced the rebound spike frequency. We further found that approximately 58% of substantia gelatinosa neurons examined expressed Ih, in which most of them were tonically firing. CONCLUSIONS: Our studies demonstrate that lidocaine strongly inhibits Ih in a reversible and concentration-dependent manner in substantia gelatinosa neurons, independent of tetrodotoxin-sensitive sodium channels. Thus, our study provides new insight into the mechanism underlying the central analgesic effect of the systemic administration of lidocaine.

Regulation of excitability in tonic firing substantia gelatinosa neurons of the spinal cord by small-conductance Ca(2+)-activated K(+) channels.

The excitability of substantia gelatinosa (SG) neurons in the spinal dorsal horn determines the processing of nociceptive information from the periphery to the central nervous system. Small conductance Ca(2+)-activated K(+) (SK) channels on neurons supply strong negative feedback control on neuronal excitability by affecting afterhyperpolarization (AHP). However, the role of SK channels in regulating tonic-firing SG neuron excitability remains elusive. In the present study, whole-cell recordings were conducted in SG neurons from acute spinal cord slices of adult rats. The SK channel opener 1-ethyl-2-benzimidazolinone (1-EBIO) attenuated spike discharges and increased AHP amplitudes; this effect was mimicked by a high Ca(2+) external solution. Systemic administration of 1-EBIO attenuated the thermal-induced nociception behavior. Conversely, the inhibition of SK channels with apamin, a specific SK channel inhibitor, increased neuronal excitability and decreased the AHP amplitudes; this effect was mimicked by a Ca(2+)-free external solution. Apamin increased excitatory synaptic transmission by increasing the amplitudes of evoked excitatory postsynaptic potentials (eEPSPs). This facilitation depended on N-methyl-d-aspartate (NMDA) receptors, extracellular Mg(2+) and intracellular Ca(2+). Voltage-gated Ca(2+) channels (VGCCs) were also involved in the apamin-induced effects. Strikingly, 1-EBIO action on decreasing excitability persisted in the presence of apamin, indicating that 1-EBIO manipulates SK channels via a pathway rather than via apamin-sensitive SK channels. The data reveal a previously uncharacterized mechanism for manipulating SG neuronal excitability by Ca(2+) conductances via both apamin-sensitive and apamin-insensitive pathways. Because SG neurons in the dorsal horn are involved in regulating nociception, manipulating neuronal excitability via SK channels indicates a potential therapeutic target.

Action of thymol on spontaneous excitatory transmission in adult rat spinal substantia gelatinosa neurons.

Thymol, which is contained in thyme essential oil, has various actions including antinociception and nerve conduction inhibition. Although thymol activates transient receptor potential (TRP) channels expressed in heterologous cells, it remains to be examined whether this is so in native neurons. It has not yet been examined how thymol affects synaptic transmission. In order to know how thymol modulates excitatory transmission with a focus on TRP activation, we investigated its effect on glutamatergic spontaneous excitatory transmission in lamina II (substantia gelatinosa; SG) neurons with which nerve terminals expressing TRP channels make synaptic contacts. The experiment was performed by using the blind whole-cell patch-clamp technique in adult rat spinal cord slices. Superfusing thymol (1 mM) for 3 min reversibly increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) with a minimal increase in its amplitude in all neurons examined. Seventy-seven% of the neurons produced an outward current at a holding potential of -70 mV. The sEPSC frequency increase and outward current produced by thymol were concentration-dependent with almost the same half-maximal effective concentration (EC50) values of 0.18 and 0.14 mM, respectively. These activities were repeated at a time interval of 30 min, although the sEPSC frequency increase but not outward current recovered with a slow time course. Voltage-gated Na(+)-channel blocker tetrodotoxin did not affect the thymol activities. The sEPSC frequency increase was inhibited by TRPA1 antagonist HC-030031 but not TRPV1 and TRPM8 antagonist (capsazepine and BCTC, respectively), while these antagonists had no effect on the outward current. This was so, albeit the two thymol activities had similar EC50 values. It is concluded that thymol increases the spontaneous release of L-glutamate onto SG neurons by activating TRPA1 channels while producing an outward current without TRP activation. Considering that the SG plays a pivotal role in modulating nociceptive transmission from the periphery, these actions of thymol could contribute to at least a part of its antinociceptive effect.

Orexin-A modulates excitatory synaptic transmission and neuronal excitability in the spinal cord substantia gelatinosa.

Although intrathecal orexin-A has been known to be antinociceptive in various pain models, the role of orexin-A in antinociception is not well characterized. In the present study, we examined whether orexin-A modulates primary afferent fiber-mediated or spontaneous excitatory synaptic transmission using transverse spinal cord slices with attached dorsal root. Bath-application of orexin-A (100nM) reduced the amplitude of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of Aδ- or C-primary afferent fibers. The magnitude of reduction was much larger for EPSCs evoked by polysynaptic C-fibers than polysynaptic Aδ-fibers, whereas it was similar in EPSCs evoked by monosynaptic Aδ- or C-fibers. SB674042, an orexin-1 receptor antagonist, but not EMPA, an orexin-2 receptor antagonist, significantly inhibited the orexin-A-induced reduction in EPSC amplitude from mono- or polysynaptic Aδ-fibers, as well as from mono- or polysynaptic C-fibers. Furthermore, orexin-A significantly increased the frequency of spontaneous EPSCs but not the amplitude. This increase was almost completely blocked by both SB674042 and EMPA. On the other hand, orexin-A produced membrane oscillations and inward currents in the SG neurons that were partially or completely inhibited by SB674042 or EMPA, respectively. Thus, this study suggests that the spinal actions of orexin-A underlie orexin-A-induced antinociceptive effects via different subtypes of orexin receptors.

Effects of tramadol on substantia gelatinosa neurons in the rat spinal cord: an in vivo patch-clamp analysis.

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. However, the precise mode of modulation remains unclear. We used an in vivo patch clamp technique in urethane-anesthetized rats to determine the antinociceptive mechanism of tramadol. 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 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 increased the sIPSCs in SG neurons. These effects were not observed following naloxone pretreatment. 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. 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). 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. M1 may play a key role in the antinociceptive mechanisms of tramadol.

Minocycline inhibits hyperpolarization-activated currents in rat substantia gelatinosa neurons.

Minocycline is a widely used glial activation inhibitor that could suppress pain-related behaviors in a number of different pain animal models, yet, its analgesic mechanisms are not fully understood. Hyperpolarization-activated cation channel-induced Ih current plays an important role in neuronal excitability and pathological pain. In this study, we investigated the possible effect of minocycline on Ih of substantia gelatinosa neuron in superficial spinal dorsal horn by using whole-cell patch-clamp recording. We found that extracellular minocycline rapidly decreases Ih amplitude in a reversible and concentration-dependent manner (IC50 = 41 µM). By contrast, intracellular minocycline had no effect. Minocycline-induced inhibition of Ih was not affected by Na(+) channel blocker tetrodotoxin, glutamate-receptor antagonists (CNQX and D-APV), GABAA receptor antagonist (bicuculine methiodide), or glycine receptor antagonist (strychnine). Minocycline also caused a negative shift in the activation curve of Ih, but did not alter the reversal potential. Moreover, minocycline slowed down the inter-spike depolarizing slope and produced a robust decrease in the rate of action potential firing. Together, these results illustrate a novel cellular mechanism underlying minocycline's analgesic effect by inhibiting Ih currents of spinal dorsal horn neurons.

Spontaneous L-glutamate release enhancement in rat substantia gelatinosa neurons by (-)-carvone and (+)-carvone which activate different types of TRP channel.

Transient receptor potential (TRP) channels in the spinal dorsal horn lamina II (substantia gelatinosa; SG), which are involved in the modulation of nociceptive transmission, have not yet been fully examined in property. Activation of the TRP channels by various plant-derived chemicals results in an increase in the spontaneous release of L-glutamate onto the SG neurons. We examined the effects of a monoterpene ketone (-)-carvone (contained in spearmint) and its stereoisomer (+)-carvone (in caraway) on glutamatergic spontaneous excitatory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. (-)-Carvone and (+)-carvone increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) in a reversible and concentration-dependent manner with a small increase in its amplitude. Half-maximal effective concentrations of (-)-carvone and (+)-carvone in increasing sEPSC frequency were 0.70 mM and 0.72 mM, respectively. The (-)-carvone but not (+)-carvone activity was inhibited by a TRPV1 antagonist capsazepine. On the other hand, the (+)-carvone but not (-)-carvone activity was inhibited by a TRPA1 antagonist HC-030031. These results indicate that (-)-carvone and (+)-carvone activate TRPV1 and TRPA1 channels, respectively, resulting in an increase in spontaneous L-glutamate release onto SG neurons, with almost the same efficacy. Such a difference in TRP activation between the stereoisomers may serve to know the properties of TRP channels in the SG.

Contribution of persistent sodium currents to the excitability of tonic firing substantia gelatinosa neurons of the rat.

The roles of persistent Na(+) currents (INaP) in intrinsic membrane properties were examined in rat substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis using a conventional whole-cell patch clamp technique. In a voltage-clamp mode, riluzole inhibited the slow voltage ramp-induced INaP but had little effect on the peak amplitude of transient Na(+) currents in SG neurons. In a current-clamp mode, most SG neurons exhibited spontaneous action potentials and tonic firing pattern. Riluzole reduced both spontaneous and elicited action potentials in a concentration-dependent manner. The present results suggest that the riluzole-sensitive INaP plays an important role in the excitability of SG neurons and are thus, likely to contribute to the modulation of nociceptive transmission from the orofacial tissues.