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.

GABA- and Glycine-Mimetic Responses of Linalool on the Substantia Gelatinosa of the Trigeminal Subnucleus Caudalis in Juvenile Mice: Pain Management through Linalool-Mediated Inhibitory Neurotransmission.

Linalool, a major odorous constituent in essential oils extracted from lavender, is known to have a wide range of physiological effects on humans including pain management. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is involved in transmission of orofacial nociceptive responses through thin myelinated A[Formula: see text] and unmyelinated C primary afferent fibers. Up to date, the orofacial antinociceptive mechanism of linalool concerning SG neurons of the Vc has not been completely clarified yet. To fill this knowledge gap, whole-cell patch-clamp technique was used in this study to examine how linalool acted on SG neurons of the Vc in mice. Under a high chloride pipette solution, non-desensitizing and repeatable linalool-induced inward currents were preserved in the presence of tetrodotoxin (a voltage-gated Na[Formula: see text]channel blocker), CNQX (a non-NMDA glutamate receptor antagonist), and DL-AP5 (an NMDA receptor antagonist). However, linalool-induced inward currents were partially suppressed by picrotoxin (a GABA[Formula: see text] receptor antagonist) or strychnine (a glycine receptor antagonist). These responses were almost blocked in the presence of picrotoxin and strychnine. It was also found that linalool exhibited potentiation with GABA- and glycine-induced responses. Taken together, these data show that linalool has GABA- and glycine-mimetic effects, suggesting that it can be a promising target molecule for orofacial pain management by activating inhibitory neurotransmission in the SG area of the Vc.

Isoflurane decreases substantia gelatinosa neuron excitability and synaptic transmission from periphery in the rat spinal dorsal horn.

Isoflurane is an inhaled anesthetic, though its actions at the cellular level remain controversial. By using acute spinal cord slices from adult rats and the whole-cell recording technique, we found that aqueous isoflurane at the minimum alveolar concentration decreased postsynaptic neural excitability and enhanced membrane conductance, while suppressing glutamate release from presynaptic afferent onto substantia gelatinosa (lamina II) neurons in the dorsal horn. The data demonstrate that isoflurane modulates synaptic transmission from peripheral to the spinal cord via both pre- and postsynaptic effects and these actions may underlie its spinal anesthesia.

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.

Potentiation of the Glycine Response by Bisphenol A, an Endocrine Disrupter, on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice.

Lamina II, also called the substantia gelatinosa (SG) of the medullary dorsal horn (the trigeminal subnucleus caudalis, Vc), is thought to play an essential role in the control of orofacial nociception because it receives the nociceptive signals from primary afferents, including thin myelinated Aδ- and unmyelinated C-fibers. Glycine, the main inhibitory neurotransmitter in the central nervous system, plays an essential role in the transference of nociceptive messages from the periphery to higher brain regions. Bisphenol A (BPA) is reported to alter the morphological and functional characteristics of neuronal cells and to be an effector of a great number of ion channels in the central nervous system. However, the electrophysiological effects of BPA on the glycine receptors of SG neurons in the Vc have not been well studied. Therefore, in this study, we used the whole-cell patch-clamp technique to determine the effect of BPA on the glycine response in SG neurons of the Vc in male mice. We demonstrated that in early neonatal mice (0-3 postnatal day mice), BPA did not affect the glycine-induced inward current. However, in the juvenile and adult groups, BPA enhanced the glycine-mediated responses. Heteromeric glycine receptors were involved in the modulation by BPA. The interaction between BPA and glycine appears to have a significant role in regulating transmission in the nociceptive pathway.

Chronic pain models amplify transient receptor potential vanilloid 1 (TRPV1) receptor responses in adult rat spinal dorsal horn.

Persistent pain is associated with negative affect originating from hypersensitivity and/or allodynia. The spinal cord is a key area for nociception as well as chronic pain processing. Specifically, the dorsal horn neurons in lamina II (substantia gelatinosa: SG) receive nociceptive inputs from primary afferents such as C fibers and/or Aδ fibers. Transient receptor potential vanilloid 1 (TRPV1) is a major receptor to sense heat as well as nociception. TRPV1 are expressed in the periphery and the central axon terminals of C fibers and/or Aδ fibers in the spinal cord. Activating TRPV1 enhances the release of glutamate in the spinal cord from naïve rodents. Here, we studied whether or not chronic pain could alter the response of TRPV1 channels to exogenous, capsaicin through study of synaptic transmission and neural activity in rat SG neurons. Using in vitro whole-cell patch-clamp recording, we found that bath application of capsaicin facilitated both the frequency and amplitude of miniature and spontaneous excitatory postsynaptic currents beyond a nerve injury and a complete Freund's adjuvant injection observed in the naïve group. Strikingly, capsaicin produced larger amplitudes of inward currents in pain models than compared to the naïve group. By contrast, the proportions of neurons that show capsaicin-induced inward currents were similar among naïve and pain groups. Importantly, the capsaicin-induced inward currents were conducted by TRPV1 and required calcium influx that was independent of voltage-gated calcium channels. Our study provides fundamental evidence that chronic inflammation and neuropathic pain models amplify the release of glutamate through the activation of TRPV1 in central axon terminals, and that facilitation of TRPV1 function in rat spinal SG neurons may contribute to enhanced capsaicin-induced inward currents.

Involvement of metabotropic glutamate receptor 5 in ethanol regulation of NMDA receptor activity in rat substantia gelatinosa neurons.

AIMS: Glutamatergic receptors are important targets of ethanol. Intake of ethanol may produce analgesic effects. The present study examined the effects of ethanol on the activity of ionotropic glutamate receptors in spinal cord substantia gelatinosa (SG) neurons, critical neurons involved in nociceptive transmission. MAIN METHODS: Whole-cell recordings were made from SG neurons of the lumbar spinal cord slices from 15 to 20-day-old rats. Ethanol and glutamate receptor agonists or antagonists were applied by superfusion. KEY FINDING: Ethanol (50 and 100 mM) applied by superfusion for 5 min dose-dependently decreased the amplitude of evoked excitatory postsynaptic potential in SG neurons. Superfusion of ethanol (100 mM) for 15 min consistently inhibited NMDA- or AMPA-induced depolarizations in SG neurons. Ethanol (100 mM) also inhibited the depolarizations induced by glutamate. However, ethanol inhibition of glutamate-induced responses significantly decreased at 10-15 min following continuous superfusion, suggesting the development of acute tolerance to the inhibition during prolonged exposure. Application of MPEP hydrochloride (an antagonist of metabotropic glutamate receptor [mGluR] 5) or GF109203X (a protein kinase C [PKC] inhibitor), together with ethanol significantly blocked the tolerance. The inhibition by ethanol of the NMDA-induced, but not AMPA-induced, depolarizations significantly decreased at 15 min during continuous superfusion while ACPD (a mGluR agonist) was co-applied with ethanol. SIGNIFICANCE: The results suggest that (1) ethanol exposure may inhibit ionotropic glutamate receptor-mediated neurotransmission; (2) regulation of NMDA receptor function by mGluR5/PKC pathways may be involved in the development of the tolerance to ethanol inhibition of glutamate-induced responses during prolonged exposure in SG neurons.

Action of Norepinephrine on Lamina X of the Spinal Cord.

Lamina X is localized in the spinal cord within the region surrounding the central canal and receives descending projections from the supraspinal nuclei. Norepinephrine (NE) is a neurotransmitter in descending pathways emanating from the brain stem; NE-containing fibers terminate in the spinal dorsal cord, particularly in the substantia gelatinosa (SG). NE enhances inhibitory synaptic transmission in SG neurons by activating presynaptic α1-receptors and hyperpolarizes the membranes of SG neurons by acting on α2-receptors; NE may thus act directly on SG neurons of the dorsal spinal cord and inhibit nociceptive transmission at the spinal level. NE-containing fibers also reportedly terminate in lamina X, suggesting that NE also modulates synaptic transmission in lamina X. However, the cellular mechanisms underlying such action have not been investigated. We hypothesized that NE might directly act on lamina X and enhance inhibitory synaptic transmission therein. Using rat spinal cord slices and in vitro whole-cell patch-clamps, we found that the bath-application of NE to lamina X does not affect the excitatory interneurons but enhances GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and induces an outward current. NE-induced enhancement of mIPSCs was blocked by α1A-receptor antagonists, and NE-induced outward current was blocked by α2-receptor antagonists. NE did not affect GABA- or glycine- induced outward currents. These findings are similar to those obtained from SG neurons: NE may act at presynaptic terminals of GABAergic and glycinergic interneurons on lamina X to facilitate inhibitory-transmitter release through α1A-receptor activation and directly induce inhibitory interneuron membrane hyperpolarization through α2-receptors activation.

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.

Moieties of plant-derived compounds responsible for outward current production and TRPA1 activation in rat spinal substantia gelatinosa.

BACKGROUND: Transient receptor potential ankyrin-1 (TRPA1) channels expressed in the central terminal of dorsal root ganglion neurons in the spinal substantia gelatinosa (SG) play a role in modulating nociceptive transmission. Although plant-derived compounds exhibiting antinociception (such as eugenol, carvacrol and thymol) activate TRPA1 channels to enhance spontaneous excitatory transmission while hyperpolarizing membranes in SG neurons without TRPA1 activation, specific chemical moieties involved in synaptic modulation are unknown. METHODS: We examined the effects of other plant-derived compounds (guaiacol, vanillin, vanillic acid and p-cymene) on holding current and spontaneous excitatory transmission at -70 mV by applying the whole-cell patch-clamp technique to SG neurons in adult rat spinal cord slices. RESULTS: None of the compounds affected the frequency or amplitude of spontaneous excitatory postsynaptic current. Guaiacol and vanillic acid had no effect on holding currents, while vanillin and p-cymene produced an inward and outward current, respectively, in some neurons tested. Synaptic modulation was also observed within the same neuron as the activities of eugenol, carvacrol, thymol, and the chemically-related plant-derived compound zingerone occurred. CONCLUSION: A substituted group in eugenol and zingerone, but not in guaiacol, vanillin or vanillic acid, as well as an OH bound to the benzene ring of carvacrol and thymol, but not p-cymene, play a role in producing outward current and TRPA1 activation. Thus, the binding of such chemical moeties to the benzene ring of plant-derived compounds appears necessary to modulate nociceptive transmission in the SG. This information provides insight for the development of new analgesics based on plant-derived compounds.

The endogenous agonist, ß-alanine, activates glycine receptors in rat spinal dorsal neurons.

ß-alanine is a structural analog of glycine and γ-aminobutyric acid (GABA) and is thought to be involved in the modulation of nociceptive information at the spinal cord. However, it is not known whether ß-alanine exerts its effect in substantia gelatinosa (SG) neurons of the spinal dorsal horn, where glycine and GABA play an important role in regulating nociceptive transmission from the periphery. Here, we investigated the effects of ß-alanine on inhibitory synaptic transmission in adult rat SG neurons using whole-cell patch-clamp. ß-alanine dose-dependently induced outward currents in SG neurons. Current-voltage plots revealed a reversal potential at approximately -70 mV, which was close to the equilibrium potential of Cl-. Pharmacological analysis revealed that ß-alanine activates glycine receptors, but not GABAA receptors. These results suggest that ß-alanine hyperpolarizes the membrane potential of SG neurons by activating Cl- channels through glycine receptors. Our findings raise the possibility that ß-alanine may modulate pain sensation through glycine receptors.

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.

P2X7 receptor-sensitivity of astrocytes and neurons in the substantia gelatinosa of organotypic spinal cord slices of the mouse depends on the length of the culture period.

The whole-cell patch-clamp technique was used to record current responses to AMPA, N-methyl-d-aspartate (NMDA), muscimol and dibenzoyl-ATP (Bz-ATP) in superficial (reactive/gliotic) substantia gelatinosa (SG) astrocytes and neurons of spinal cord slices kept for different periods of time in organotypic culture. Currents induced by AMPA, NMDA and muscimol confirmed the existence of their specific receptors in 2-week-old neurons; astrocytes cultured for the same period of time responded to AMPA and muscimol, but not to NMDA. AMPA had a larger effect on 2-week-old astrocytes than on the 1-week-old ones, in spite of a similar sensitivity of the age-matched neurons to this amino acid. The effect of the prototypic P2X7 receptor agonist Bz-ATP on superficial astrocytes and neurons depended on the drug concentration applied and increased in parallel with the lengthening of the culture period. The amplitudes of Bz-ATP currents of deep (resting) astrocytes were age-independent. Neurons located in deep layers exhibited after 1week of culturing much larger Bz-ATP currents than the superficial ones of the same age. In conclusion, whereas resting astrocytes had culture period-independent P2X7 receptor-sensitivity, reactive/gliotic astrocytes exhibited P2X7 receptor-sensitivity increasing in parallel with the prolongation of the time spent in culture. The results with Bz-ATP agree with the facilitation of AMPA-induced currents in reactive astrocytes during development, and with the hypothesis that extracellular ATP is an ontogenetically early transmitter/signaling molecule in the CNS.

Presynaptic facilitation by tetracaine of glutamatergic spontaneous excitatory transmission in the rat spinal substantia gelatinosa - Involvement of TRPA1 channels.

The amide-type local anesthetic (LA) lidocaine activates transient receptor potential (TRP) ankyrin-1 (TRPA1) channels to facilitate spontaneous l-glutamate release onto spinal substantia gelatinosa (SG) neurons, which play a crucial role in regulating nociceptive transmission. In contrast, the ester-type LA procaine reduces the spontaneous release of l-glutamate in SG neurons. In order to determine whether TRPA1 activation by LAs is specific to amide-types, we examined the actions of tetracaine, another ester-type LA, and other amide-type LAs on glutamatergic spontaneous excitatory transmission in SG neurons by focusing on TRP activation. Whole-cell patch-clamp recordings were performed on SG neurons of adult rat spinal cord slices at a holding potential of -70mV. Bath-applied tetracaine increased spontaneous excitatory postsynaptic current (sEPSC) frequency in a concentration-dependent manner. Tetracaine activity was resistant to the voltage-gated Na+-channel blocker tetrodotoxin, the TRP vanilloid-1 antagonist capsazepine, and the TRP melastatin-8 antagonist BCTC, but was inhibited by the non-selective TRP antagonist ruthenium red and the TRPA1 antagonist HC-030031. With respect to amide-type LAs, prilocaine had a tendency to increase sEPSC frequency, while ropivacaine and levobupivacaine reduced the frequency. In conclusion, tetracaine facilitated spontaneous l-glutamate release from nerve terminals by activating TRPA1 channels in the SG, resulting in an increase in the excitability of SG neurons. TRPA1 activation was not specific to amide-type or ester-type LAs. The facilitatory action of LAs may be involved in pain occurring after recovery from spinal anesthesia.

Effects of hypotaurine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in immature mice.

To understand the action and mechanism of hypotaurine, an immediate precursor of taurine, on orofacial nociceptive processing, we examined the direct effects and receptor types involved in hypotaurine-induced responses using the whole-cell patch clamp technique in the substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) of immature mice. Under the condition of high-chloride pipette solution, hypotaurine elicited inward currents or upward deflections of membrane potential, which increased in a concentration-dependent manner (30-3000 µM) with the EC50 of 663.8 and 337.6 µM, respectively. The responses to 300 µM hypotaurine were reproducible and recovered upon washout. The 300 µM hypotaurine-induced currents were maintained in the presence of TTX, CNQX, and AP5, indicating direct postsynaptic action of hypotaurine on SG neurons. Responses to both low (300 µM) and high (1 or 3 mM) concentrations of hypotaurine were completely and reversibly blocked by the glycine receptor antagonist strychnine (2 µM), but unaffected by the GABAA receptor antagonist gabazine (3 µM) which blocks synaptic GABAA receptors at low concentration. Furthermore, responses to 300 µM hypotaurine and a maximal concentration of glycine (3 mM) were not additive, indicating that hypotaurine and glycine act on the same receptor. Hypotaurine-induced currents were partially antagonized by picrotoxin (50 µM) which blocks homomeric glycine receptors and by bicuculline (10 µM) which is an antagonist of α2 subunit-containing glycine receptors. These results suggest that hypotaurine-induced responses were mediated by glycine receptor activation in the SG neurons and hypotaurine might be used as an effective therapeutics for orofacial pain.

The role of Cx36 and Cx43 in 4-aminopyridine-induced rhythmic activity in the spinal nociceptive dorsal horn: an electrophysiological study in vitro.

Connexin (Cx) proteins and gap junctions support the formation of neuronal and glial syncytia that are linked to different forms of rhythmic firing and oscillatory activity in the CNS. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to profile developmental expression of two specific Cx proteins, namely glial Cx43 and neuronal Cx36, in postnatal lumbar spinal cord aged 4, 7, and 14 days. Extracellular electrophysiology was used to determine the contribution of Cx36 and Cx43 to a previously described form of 4-aminopyridine (4-AP)-induced 4-12 Hz rhythmic activity within substantia gelatinosa (SG) of rat neonatal dorsal horn (DH) in vitro. The involvement of Cx36 and Cx43 was probed pharmacologically using quinine, a specific uncoupler of Cx36 and the mimetic peptide blocker Gap 26 which targets Cx43. After establishment of 4-12 Hz rhythmic activity by 4-AP (25 µmol/L), coapplication of quinine (250 µmol/L) reduced 4-AP-induced 4-12 Hz rhythmic activity (P < 0.05). Preincubation of spinal cord slices with Gap 26 (100 µmol/L), compromised the level of 4-AP-induced 4-12 Hz rhythmic activity in comparison with control slices preincubated in ACSF alone (P < 0.05). Conversely, the nonselective gap junction "opener" trimethylamine (TMA) enhanced 4-12 Hz rhythmic behavior (P < 0.05), further supporting a role for Cx proteins and gap junctions. These data have defined a physiological role for Cx36 and Cx43 in rhythmic firing in SG, a key nociceptive processing area of DH. The significance of these data in the context of pain and Cx proteins as a future analgesic drug target requires further study.

Selective 5-HT7 receptor agonists LP 44 and LP 211 elicit an analgesic effect on formalin-induced orofacial pain in mice.

OBJECTIVE: To investigate the antinociceptive effects of pharmacological activation of 5-HT7 receptors on orofacial pain in mice. MATERIAL AND METHODS: Nociception was evaluated by using an orofacial formalin test in male Balb-C mice. Selective 5-HT7 receptor agonists, LP 44 and LP 211 (1, 5, and 10 mg/kg), were given intraperitoneally 30 min prior to a formalin injection. A bolus of 10 µl of 4% subcutaneous formalin was injected into the upper lip of mice and facial grooming behaviors were monitored. The behavioral responses consisted of two distinct periods, the early phase corresponding to acute pain (Phase I: 0-12 min) and the late phase (Phase II: 12-30 min). RESULTS: LP 44 and LP 211 (1, 5, and 10 mg/kg) produced an analgesic effect with reductions in face rubbing time in both Phase I and Phase II of the formalin test. CONCLUSION: Our results suggest that 5-HT7 receptor agonists may be promising analgesic drugs in the treatment of orofacial pain.

Selective 5-HT7 receptor agonists LP 44 and LP 211 elicit an analgesic effect on formalin-induced orofacial pain in mice

ABSTRACT The most recently identified serotonin (5-HT) receptor is the 5-HT7 receptor. The antinociceptive effects of a 5-HT7 receptor agonist have been shown in neuropathic and inflammatory animal models of pain. A recent study demonstrated the functional expression of 5-HT7 receptors in the substantia gelatinosa (SG) of the trigeminal subnucleus caudalis, which receives and processes orofacial nociceptive inputs. Objective To investigate the antinociceptive effects of pharmacological activation of 5-HT7 receptors on orofacial pain in mice. Material and Methods Nociception was evaluated by using an orofacial formalin test in male Balb-C mice. Selective 5-HT7 receptor agonists, LP 44 and LP 211 (1, 5, and 10 mg/kg), were given intraperitoneally 30 min prior to a formalin injection. A bolus of 10 µl of 4% subcutaneous formalin was injected into the upper lip of mice and facial grooming behaviors were monitored. The behavioral responses consisted of two distinct periods, the early phase corresponding to acute pain (Phase I: 0–12 min) and the late phase (Phase II: 12–30 min). Results LP 44 and LP 211 (1, 5, and 10 mg/kg) produced an analgesic effect with reductions in face rubbing time in both Phase I and Phase II of the formalin test. Conclusion Our results suggest that 5-HT7 receptor agonists may be promising analgesic drugs in the treatment of orofacial pain.