Hai-Yuan Yue

Effect of resiniferatoxin on glutamatergic spontaneous excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord

The transient receptor potential (TRP) vanilloid type 1 (TRPV1) agonist, capsaicin, enhances glutamatergic spontaneous excitatory synaptic transmission in CNS neurons. Resiniferatoxin (RTX) has a much higher affinity for TRPV1 than capsaicin, but its ability to modulate excitatory transmission is unclear. We examined the effect of RTX on excitatory transmission using the whole-cell patch-clamp technique in substantia gelatinosa (SG) neurons of adult rat spinal cord slices. Bath-applied RTX dose-dependently increased the frequency, but not the amplitude, of spontaneous excitatory postsynaptic current (sEPSC), independent of its application time. In about a half of the neurons tested, this effect was accompanied by an inward current at -70 mV that was sensitive to glutamate-receptor antagonists. Repeated application of RTX did not affect excitatory transmission. RTX was more potent than capsaicin but showed similar efficacy. RTX activity could be blocked by capsazepine or SB-366791, a TRPV1 antagonist, but not tetrodotoxin, a Na(+)-channel blocker, and could be inhibited by pretreatment with capsaicin but not the TRPA1 agonist, allyl isothiocyanate. RTX enhances the spontaneous release of L-glutamate from nerve terminals with similar efficacy as capsaicin and produces a membrane depolarization by activating TRPV1 in the SG, with fast desensitization and slow recovery from desensitization. These results indicate a mechanism by which RTX can modulate excitatory transmission in SG neurons to regulate nociceptive transmission.

Phospholipase A2 activation by melittin enhances spontaneous glutamatergic excitatory transmission in rat substantia gelatinosa neurons.

In order to know a role of phospholipase A2 in modulating nociceptive transmission, the effect of a secreted phospholipase A2 activator melittin on spontaneous glutamatergic excitatory transmission was investigated in substantia gelatinosa neurons of an adult rat spinal cord slice by using the whole-cell patch-clamp technique. Bath-applied melittin at concentrations higher than 0.5 microM increased both the amplitude and the frequency of spontaneous excitatory postsynaptic current in a manner independent of tetrodotoxin; the latter effect of which was examined in detail. In 80% of the neurons examined (n = 64), melittin superfused for 3 min gradually increased spontaneous excitatory postsynaptic current frequency (by 65+/-6% at 1 microM; n = 51) in a dose-dependent manner (effective concentration for half-maximal effect = 1.1 microM). This effect subsided within 3 min after washout. The spontaneous excitatory postsynaptic current frequency increase produced by melittin was reduced by the phospholipase A2 inhibitor 4-bromophenacryl bromide (10 microM) while being unaffected by the cyclooxygenase inhibitor indomethacin (100 microM) and the lipoxygenase inhibitor nordihydroguaiaretic acid (100 microM). A similar increase in spontaneous excitatory postsynaptic current frequency was produced by exogenous arachidonic acid (50 microM); this effect was also unaffected by the cyclooxygenase or lipoxygenase inhibitor. Melittin failed to increase spontaneous excitatory postsynaptic current frequency in a nominally Ca2+-free or La3+-containing Krebs solution. We conclude that melittin increases the spontaneous release of L-glutamate to substantia gelatinosa neurons by activating secreted phospholipase A2 and increasing Ca2+ influx through voltage-gated Ca2+ channels in nerve terminals, probably with an involvement of arachidonic acid but not its metabolites produced by cyclooxygenase and lipoxygenase. Considering that the substantia gelatinosa plays an important role in regulating nociceptive transmission, it is suggested that this transmission may be positively modulated by secreted phospholipase A2 activation in the substantia gelatinosa.

TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase

We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1-5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na(+)-channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of L-glutamate.

AM404 enhances the spontaneous release of L-glutamate in a manner sensitive to capsazepine in adult rat substantia gelatinosa neurones

In 84% of substantia gelatinosa (SG) neurones examined in adult rat spinal cord slices, an anandamide transport inhibitor, AM404, increased the frequency of spontaneous excitatory postsynaptic currents in a manner similar to that of capsaicin. AM404 was without actions in the presence of a vanilloid TRPV1 receptor antagonist, capsazepine. We conclude that AM404 enhances the spontaneous release of L-glutamate by activating TRPV1 receptors in the SG.

Biphasic modulation by galanin of excitatory synaptic transmission in substantia gelatinosa neurons of adult rat spinal cord slices

Although intrathecally administrated galanin modulates nociceptive transmission in a biphasic manner, this has not been fully examined previously. In the present study, the action of galanin on synaptic transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices was examined, using the whole cell patch-clamp technique. Galanin concentration-dependently increased the frequency of spontaneous excitatory postsynaptic current (EPSC; EC(50) = 2.0 nM) without changing the amplitude, indicating a presynaptic effect. This effect was reduced in a Ca(2+)-free, or voltage-gated Ca(2+) channel blocker La(3+)-containing Krebs solution and was produced by a galanin type-2/3 receptor (GalR2/R3) agonist, galanin 2-11, but not by a galanin type-1 receptor (GalR1) agonist, M617. Galanin also concentration-dependently produced an outward current at -70 mV (EC(50) = 44 nM), although this appeared to be contaminated by a small inward current. This outward current was mimicked by M617, but not by galanin 2-11. Moreover, galanin reduced monosynaptic Aδ-fiber- and C-fiber-evoked EPSC amplitude; the former reduction was larger than the latter. A similar action was produced by galanin 2-11, but not by M617. Spontaneous and focally evoked inhibitory (GABAergic and glycinergic) transmission was unaffected by galanin. These findings indicate that galanin at lower concentrations enhances the spontaneous release of l-glutamate from nerve terminals by Ca(2+) entry from the external solution following GalR2/R3 activation, whereas galanin at higher concentrations also produces a membrane hyperpolarization by activating GalR1. Moreover, galanin reduces l-glutamate release onto SG neurons from primary afferent fibers by activating GalR2/R3. These effects could partially contribute to the behavioral effect of galanin.

TRPV1 agonist piperine but not olvanil enhances glutamatergic spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at -70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 μM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.

Zingerone enhances glutamatergic spontaneous excitatory transmission by activating TRPA1 but not TRPV1 channels in the adult rat substantia gelatinosa

Transient receptor potential (TRP) channels are thought to play a role in regulating nociceptive transmission to spinal substantia gelatinosa (SG) neurons. It remains to be unveiled whether the TRP channels in the central nervous system are different in property from those involved in receiving nociceptive stimuli in the peripheral nervous system. We examined the effect of the vanilloid compound zingerone, which activates TRPV1 channels in the cell body of a primary afferent neuron, on glutamatergic excitatory transmission in the SG neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Bath-applied zingerone reversibly and concentration-dependently increased spontaneous excitatory postsynaptic current (EPSC) frequency. This effect was accompanied by an inward current at -70 mV that was resistant to glutamate receptor antagonists. These zingerone effects were repeated and persisted in Na(+)-channel blocker tetrodotoxin-, La(3+)-, or IP3-induced Ca(2+)-release inhibitor 2-aminoethoxydiphenyl borate-containing or Ca(2+)-free Krebs solution. Zingerone activity was resistant to the selective TRPV1 antagonist capsazepine but sensitive to the nonselective TRP antagonist ruthenium red, the TRPA1 antagonist HC-030031, and the Ca(2+)-induced Ca(2+)-release inhibitor dantrolene. TRPA1 agonist allyl isothiocyanate but not capsaicin inhibited the facilitatory effect of zingerone. On the other hand, zingerone reduced monosynaptically evoked EPSC amplitudes, as did TRPA1 agonists. Like allyl isothiocyanate, zingerone enhanced GABAergic spontaneous inhibitory transmission in a manner sensitive to tetrodotoxin. We conclude that zingerone presynaptically facilitates spontaneous excitatory transmission, probably through Ca(2+)-induced Ca(2+)-release mechanisms, and produces a membrane depolarization in SG neurons by activating TRPA1 but not TRPV1 channels.

Oxytocin produces a membrane depolarization in adult rat spinal substantia gelatinosa neurons

The development of pain after peripheral nerve and tissue injury involves not only neuronal pathways but also glia. However, uncertainty still remains as to the relative contribution of different types of glial cells in the development of the pain-related enhanced response states. We examined the contribution of glial cells to the central sensitization in the rat spinal dorsal horn which is induced in neuropathic pain and inflammatory pain. In rats subjected to neuropathic pain, the immunoreactivity (IR) of microglial marker OX 42 was largely increased. In rats subjected to inflammatory pain, IR of astosytes marker GFAP was slightly increased. The optically-recorded neuronal excitation induced by single-pulse stimulation to the dorsal root was augmented in rats subjected to neuropathic pain and rats subjected to inflammatory pain by comparison to control rats. The bath application of a microglial inhibitor minocycline and a p38 mitogen-activated protein kinase inhibitor SB203580 inhibited the neuronal excitation in rats subjected to neuropathic pain, but not in control and rats subjected to inflammatory pain. PPADS slightly inhibited the neuronal excitation in all group. The additional perfusion of TNP-ATP in PPADS largely inhibited the neuronal excitation in rats subjected to neuropathic pain. In contrast, an astroglial toxin L-alpha-aminoadipate and a gap junction blocker carbenoxolone inhibited the neuronal excitation in rats subjected to inflammatory pain, but not in control and rats subjected to neuropathic pain. The larger number of cells in the spinal cord slice taken from rats subjected to neuropathic pain showed the Ca2+ signal by puff application of ATP to comparison with control and rats subjected to inflammatory pain. The Ca2+ signal was inhibited by minocycline and TNP-ATP. Research fund: KAKENHI22600005.

Opioids reduce the peak amplitude of action potential in adult rat dorsal root ganglion neurons

GABA and glycine are important inhibitory neurotransmitters in the central nervous system and are loaded into synaptic vesicles via the vesicular GABA transporter (VGAT). Due to the evidence linking alterations in GABAergic and/or glycinergic neurotransmission to various pain disorders, we investigated the possible influence of downregulation of VGAT on pain threshold in mice. The phenotypes of heterozygous VGAT knockout (VGAT+/−) mice were compared to wild-type (WT) mice using behavioral and electrophysiological assays. Western blot analysis showed significant reduction of VGAT protein levels in VGAT+/− mice brain (47.8% of WT mice) and spinal cord (61.0% of WT mice). However, HPLC revealed that glutamate, GABA, and glycine contents in the whole brain and spinal cord were no changed in VGAT+/− mice. Although behavioral analysis of VGAT+/− mice showed unchanged motor coordination, anxiety, memory performance and anesthetic sensitivity to propofol and ketamine, thermal nociception and inflammatory pain were enhanced. Patch-clamp recordings revealed that the frequency and amplitude of glycinergic mIPSCs in dorsal horn neurons were reduced in VGAT+/− mice. Genotype differences in glycinergic mIPSCs were more evident during sustained stimulation by high potassium solutions. It seemed that the estimated size of the readily releasable pool (RRP) of glycine-containing vesicles was reduced in VGAT+/− mice. Taken together, our results provide genetic, behavioral and electrophysiological evidence that reduction of VGAT-mediated inhibitory drive in the spinal cord alters very specific forms of pain processing. Research fund: KAKENHI (40447264).