Dorsal root ganglion (DRG) neurons that innervate the skeletal muscle (SK-DRG neurons) are differentiated into various subpopulations and each exhibits different receptive properties expressing different ion channels in different combinations. In this study, we introduce a novel method for in vivo patch-clamp recording of the SK-DRG neurons in rats to allow integrated analysis of their diverse properties.
   To identify the SK-DRG neurons, the fluorescent retrograde tracer DiI was injected into the left anterior tibial muscle of 6-7-week-old rats anesthetized with pentobarbital sodium. Three to 6 weeks after the injection, the rats were deeply anesthetized again, and each animal was mounted on the stage of an upright microscope. After isolating the left L4-DRG along with the sciatic nerve, maintaining the peripheral connection, it was placed in a chamber on the stage. The individual neurons on the ganglionic surface were then visualized.Whole-cell patch-clamp recording was established from the DiI-positive DRG neurons.
   Small- and medium-sized DRG neurons, which are mainly associated with nociception, could be classified into subpopulations based on axonal conduction velocity, receptive properties (mechanical and thermal stimuli), and current expression profiles (hyperpolarization-activated current and instantaneous current). Some of them responded to experimental ischemic muscle contraction. Therefore, we discuss some components that could cause muscle pain during ischemic contraction.
   Our novel method is useful for analyzing the diverse properties of SK-DRG neurons and for ascertaining the factors that cause pain in pathological conditions such as ischemia.

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   Spinal cord electrical stimulation (SCS) is the most commonly used implantable neuro stimulation modality for the management of pain syndromes. However, cellular mechanisms of SCS-evoked analgesia have been poorly understood at this time. In the present study, whole-cell patch-clamp recordings were performed from substantia gelatinosa (SG) neurons in adult rat spinal cord slices to determine which neurotransmitters are influenced by SCS. Although repetitive stimuli applied to the dorsal root did not induce any slow responses, ones focally applied to the spinal dorsal horn produced slow inhibitory postsynaptic currents (IPSCs) at a holding potential of -50 mV in about 30% of the SG neurons recorded. The amplitude and duration of slow IPSCs increased with the number of stimuli and decreased with removal of Ca²⁺ from the external Krebs solution. Slow IPSCs were associated with an increase in membrane conductance; their polarity was reversed at a potential close to the equilibrium potential for K⁺, calculated from the Nernst equation. Slow IPSCs were blocked by addition of GDP-β-S into the patch-pipette solution, reduced in amplitude in the presence of Ba²⁺, and significantly suppressed in the presence of an antagonist of GIRK channels, tertiapin-Q. Somatostatin produced an outward current in a subpopulation of SG neurons and the slow IPSC was occluded during the somatostatin-induced outward current. Moreover, slow IPSCs were significantly inhibited by a somatostatin receptor antagonist,cyclo-somatostatin. These results indicate that endogenous somatostatin released from interneurons or descending fibers by SCS may induce slow IPSCs by activating GIRK channels in SG neurons. It is suggested that this slow synaptic transmission may play an important role in SCS-evoked analgesia.

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   Galanin, a 29 ⁄ 30 amino acid neuropeptide, and three types (GalR1-3) of receptors for this neuropeptide, which are expressed in rat dorsal root ganglion neurons and spinal dorsal horn neurons, play a pivotal role in regulating nociceptive transmission to the spinal dorsal horn from the periphery. Intrathecal administration of galanin in rats modulates nociceptive behavior in a biphasic manner such that galanin at low and high doses produces nociception and antinociception, respectively. In order to know cellular mechanisms for this effect, we investigated the effect of galanin on glutamatergic spontaneous excitatory synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by use of the whole cell patch-clamp technique. In 83% of the neurons examined (n=29), galanin (0.03 µM) superfused for 2 min increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) recorded at -70 mV without a change in its amplitude and also in holding currents. The remaining neurons produced a small outward (n=4) or inward current (n=1). This presynaptic effect was dose-dependent with an EC₅₀ value of 0.0029 µM, and was mimicked by a selective GalR2⁄R3 agonist galanin (2-11) but not a selective GalR1 agonist M617. Superfusing galanin at a higher dose such as 0.1 µM for 2 min produced an outward current in 39% of SG neurons tested (n=33). Twelve % of the SG neurons had an inward current and the remaining neurons did not change holding currents. The outward current produced by galanin had an EC₅₀ value of 0.042 µM, and was mimicked by M617 but not galanin (2-11). The sEPSC frequency increase and outward current persisted in the presence of a Na⁺-channel blocker tetrodotoxin.It is concluded that galanin at lower doses enhances the spontaneous release of L-glutamate from nerve terminals by activating GalR2⁄R3 while galanin at higher doses produces a membrane hyperpolarization by activating GalR1 in the SG. This pre- and postsynaptic effects could contribute to at least a part of the behavioral effect of galanin.

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   Negative emotion resulting from nociception is the most serious therapeutic concern especially in treatment of chronic persistent pain. The spino-parabrachio (PB)-amygdaloid pathway originating in the superficial layer of the dorsal horn and terminating in the laterocapsular part (CeLC) of the central amygdala plays a pivotal role in the expression of such painrelated negative emotion (Gauriau and Bernard, 2002). Bird et al. (2005) demonstrated that, in arthritis pain model, the PB-CeLC synaptic potentiation involves protein kinase A-dependent enhancement of NMDA receptor function. In contrast, we demonstrated that the PBCeLC transmission becomes potentiated in rats with neuropathic pain persisting for a week (Ikeda et al., 2007) in a manner not involving NMDA receptor component potentiation, unlike that in the arthritis model rats. To determine whether this difference in the mechanisms of PB-CeLC synaptic potentiation between these pain models results from that in the timecourse after operation in these experiments or in the modality of pain in these pain models,we investigated PB-CeLC synaptic potentiation in rats with incipient neuropathic pain. Excitatory postsynaptic currents (EPSC) evoked by PB afferent stimulation in CeLC neurons were evaluated in the brain slices after measurement of allodynic response with von Frey filaments in vivo at 6 - 8 hr after the nerve ligation. The allodynic response was observed only in the left-side hindpaw and evoked EPSC amplitude was potentiated in the bilateral CeLCs. This potentiation, observed in both sides of the CeLC, was primarily due to enhanced nonNMDA receptor components, in a manner similar to that observed in the same pain model with prolonged (1 week) period of pain. In conclusion, the different synaptic mechanisms underlying the PB-CeLC synaptic potentiation between arthritis and neuropathic pain models do not result from the difference in their time-course but rather from their distinct spatiotemporal patterns of central activation with distinct modalities.

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   In order to evaluate the neuronal mechanisms of abnormal pain following TMJ inflammation, the expression of Transient Receptor Potential (TRP) channels in trigeminal ganglion neurons (TRG neurons) were precisely analyzed in rats with temporomandibular joint (TMJ) inflammation.
   The facial skin temperature was significantly increased at 1 and 3 days after complete Freund's adjuvant (CFA) injection into the TMJ and that was decreased at 7 days after that. On the other hand, we could not observe any facial skin temperature changes in sham-operated rats. Many inflammation mediated cells were observed in the TMJ region at 3 days after CFA injection.Furthermore, the small number of inflammation mediated cells was distributed in the TMJ region at 7 days after CFA injection. TRPV1 mRNA was expressed in small TRG neurons (≤ 200 µm²) 3 days after CFA injection. On the other hand, the number of TRG neurons expressed TRPM8 mRNA was significantly larger at 7 days after CFA injection in 201 - 300 µm² and 401 - 500 µm² TRG cells. TRPA1 mRNA was expressed in 101 - 200 µm² TRG cells at 3 days after CFA injection.
   The present findings revealed that TRPV1 and TRPA1 in TRG cells and inflammation-medicated cells in TMJ region expressed with similar time-course associated with facial temperature change after CFA injection, suggesting that these channels are involved in the trigeminal neuropathic pain following TMJ inflammation.

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   It is well-known that capsaicin (Caps) activates TRPV1s existing in the peripheral and central terminals of primary-afferent fibers; the peripheral activation of TRPV1 produces action potentials and the central activation of TRPV1 leads to a barrage of the spontaneous release of L-glutamate from nerve terminals to spinal dorsal horn neurons. Although Caps was reported to produce a nerve conduction block, this action has not been thoroughly examined yet. We examined the actions of Caps and its analogs on Na⁺-channel blocker tetrodotoxin-sensitive and fast-conducting compound action potentials (CAPs) recorded from the frog sciatic nerve by use of the air-gap method.Caps reversibly reduced the peak amplitude of the CAP in a dose-dependent manner (by about 50% at 200 µM). Although a TRPV1 antagonist capsazepine (50 µM) by itself inhibited CAPs, this drug did not affect the Caps-induced inhibition of CAP. A TRPV1 agonist resiniferatoxin (5 µM) had no effect on CAPs. Caps analogs, dihydrocapsaicin, zingerone, eugenol and vanillin, also inhibited CAPs in a reversible and dose-dependent manner. A potency sequence of these inhibitions was Caps = dihydrocapsaicin > eugenol >> zingerone ≥ vanillin >> vanillylamine. Other Caps analog, vanillic acid, had almost no effect on CAPs. A TRPV1 agonist, olvanil, at 30 µM, a maximal concentration to dissolve this drug in Ringer solution, was less effective than was Caps in inhibiting CAPs. It is concluded that Caps inhibits CAPs without TRPV1 activation and that a chemical structure bound to the vanillyl group of Caps analogs plays a role in determining the extent of CAP inhibition. These results may serve to know molecular mechanisms for Caps-induced conduction block.

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